Ebook Netter’s essential biochemistry: Part 1

(BQ) Part 1 book Netter’s essential biochemistry hass contents: Human karyotype and the structure of DNA, basic genetics for biochemistry, cell cycle and cancer, enzymes and consequences of enzyme deficiencies, biological membranes,... and other contents.

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NETTER:

It’s How You Know Anatomy.

Netter Collection of Medical Illustrations, 2nd Edition

The Netter Collection of Medical Illustrations,

Dr Frank H Netter’s decades of work devoted to depicting each of the major body systems, has been updated and brought into modern context

The second edition of the legendary “green books” offers Netter’s timeless work, now arranged and enhanced by modern text and radiologic imaging Contributions by eld-leading doctors and teachers from world-renowned medical institutions are

supplemented with new illustrations created by master artist-physician Carlos Machado and other top medical illustrators working in the Netter tradition

Netter’s Correlative Imaging Series

The Netter’s Correlative Imaging series pairs classic Netter and Netter-style illustrations with imaging

studies and succinct descriptions to provide you with a richer understanding of human anatomy

These comprehensive, normal anatomy atlases cover all major sections of the body, featuring illustrated plates side-by-side with the most common imaging modalities for each region.

Shop online at elsevierhealth.com

Entire Collection Now Available!

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Netter’s Essential HistologyWith Student Consult Access

By William K Ovalle, PhD, and Patrick C Nahirney, PhD

Bring histologic concepts to life through beautiful Netter illustrations!

Netter’s Atlas of NeuroscienceWith Student Consult Access

By David L Felten, MD, PhD, M Kerry O’Banion, MD, PhD, and Mary Summo Maida, PhD

Master the neuroscience fundamentals needed for the classroom and beyond.

Netter’s Essential Physiology With Student Consult Access

By Susan E Mulroney, PhD, and Adam K Myers, PhD

Enhance your understanding of physiology the Netter way!

Netter’s Atlas of Human EmbryologyWith Student Consult Access

By Larry R Cochard, PhD, et al

Finally an accessible introduction to diagnostic imaging!

Netter’s Illustrated Human PathologyWith Student Consult Access

By L Maximilian Buja, MD, and Gerhard R F Krueger, PhD

Gain critical insight into the structure-function relationships and the pathological basis of human disease!

Netter’s Illustrated PharmacologyWith Student Consult Access

By Robert B Raffa, PhD, Scott M Rawls, PhD, and Elena Portyansky Beyzarov, PharmD

Take a distinct visual approach to understanding both the basic science and clinical applications of pharmacology.

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NETTER BASIC SCIENCE COLLECTION!

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NETTER’S ESSENTIAL BIOCHEMISTRY

PETER RONNER, PhDPro essor o Biochemistry and Molecular Biology

Pro essor o Pharmaceutical SciencesDepartment o Biochemistry and Molecular Biology

T omas Jef erson UniversityPhiladelphia, Pennsylvania

if any Davanzo, MA, CMI

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NE ER’S ESSEN IAL BIOCHEMIS RY ISBN: 978-1-929007-63-9

No part o this publication may be reproduced or transmitted in any orm or by any means, electronic or

mechanical, including photocopying, recording, or any in ormation storage and retrieval system, without

permission in writing rom the publisher Details on how to seek permission, urther in ormation about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be ound at our website: www.elsevier.com/permissions

T is book and the individual contributions contained in it are protected under copyright by the Publisher

(other than as may be noted herein).

Permission or Netter Art gures may be sought directly rom Elsevier’s Health Science Licensing Department

in Philadelphia, PA: phone 800-523-1649, ext 3276, or 215-239-3276; or email H.licensing@elsevier.com

Notices

Knowledge and best practice in this eld are constantly changing As new research and experience broaden our understanding, changes in research methods, pro essional practices, or medical treatment may become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any in ormation, methods, compounds, or experiments described herein In using such in ormation

or methods they should be mind ul o their own sa ety and the sa ety o others, including parties or whom they have a pro essional responsibility.

With respect to any drug or pharmaceutical products identi ed, readers are advised to check the most current in ormation provided (i) on procedures eatured or (ii) by the manu acturer o each product to be administered, to veri y the recommended dose or ormula, the method and duration o administration,

and contraindications It is the responsibility o practitioners, relying on their own experience and

knowledge o their patients, to make diagnoses, to determine dosages and the best treatment or each

individual patient, and to take all appropriate sa ety precautions.

o the ullest extent o the law, neither the Publisher nor the authors, contributors, or editors, assume any liability or any injury and/or damage to persons or property as a matter o products liability,

negligence or otherwise, or rom any use or operation o any methods, products, instructions, or ideas

contained in the material herein.

Library o Congress Cataloging-in-Publication Data

Names: Ronner, Peter, 1951- author | Netter, Frank H (Frank Henry),

1906-1991, illustrator | Machado, Carlos A G., illustrator |

Craig, John A., illustrator | Perkins, James A., illustrator.

itle: Netter’s biochemistry / Peter Ronner ; illustrations by

Frank H Netter ; contributing illustrators, Carlos A.G Machado,

John A Craig, James A Perkins.

Other titles: Biochemistry

Description: Philadelphia, PA : Elsevier, [2018] | Includes bibliographical

re erences and index.

Identi ers: LCCN 2016024484 | ISBN 9781929007639 (pbk : alk paper)

Subjects: | MESH: Biochemical Phenomena | Biochemistry

Classi cation: LCC QP514.2 | NLM QU 34 | DDC 572–dc23 LC record available at

https://lccn.loc.gov/2016024484

Executive Content Strategist: Elyse O’Grady

Content Development Specialist: Stacy Eastman

Publishing Services Manager: Patricia annian

Senior Project Manager: Carrie Stetz

Design Direction: Julia Dummitt

Printed in China

Last digit is the print number: 9 8 7 6 5 4 3 2 1

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o Wanda and Lukas

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Peter Ronner, PhD, is Pro essor o Biochemistry and Molecular Biology at the

Sidney Kimmel College o Medicine at T omas Jef erson University in delphia He holds a secondary appointment as Pro essor o Pharmaceutical Sciences in the College o Pharmacy at T omas Jef erson University Dr Ronner received his PhD in Biochemistry rom the Swiss Federal Institute o echnol-ogy (E H) in Zurich His ormer laboratory research involved studies o pan-creatic hormone secretion Dr Ronner has taught medical students or nearly

Phila-30 years and pharmacy students or almost 10 years He is also a past chair o the Association o Biochemistry Course Directors (now Association o Bio-chemistry Educators) At Jef erson, he has received numerous awards or his teaching, including a Lindback Award and a portrait painting

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Many people have helped me write this book, but Dr John

T omas rom New York University School o Medicine

deserves a place o honor He and I worked on this book

together until we were orced to pause or a ew years

Years earlier, at the University o Pennsylvania, the late Dr

Annemarie Weber introduced me to teaching biochemistry to

medical students She was a tremendous role model At

T omas Jef erson University, Dr Darwin Prockop planted in

my mind the idea o writing a biochemistry textbook Many

years later, Paul Kelly (then at Icon Learning Systems),

approached me with the idea o using Dr Netter’s images or

a biochemistry review book T is appealed to me because

biochemistry is taught as a rather abstract science that

stu-dents have di culty linking to actual patients T e Netter

images, I hoped, would provide the views o the practicing

physician T anks to the support o my chairman, Dr Jef rey

Benovic, this book project became part o my scholarly

pur-suits I am thank ul or the invaluable eedback the many

students o medicine and pharmacy at Jef erson gave me over

the years

I would like to thank the team at Elsevier or their support,

especially Elyse O’Grady (Senior Content Strategist), Stacy

Eastman and Marybeth T iel (Content Development

Special-ists), as well as Carrie Stetz (Senior Project Manager/

Specialist)

Finally, I would like to thank my amily and riends or

their support while writing this book

T is book is dedicated to my wi e Wanda and my son

Lukas Wanda has been a key in uence on me, because she

has continuously given me her perspective as a practicing

physician and medical student educator Lukas, a chemistry

major and current medical student, has been my most trusted

adviser on questions about young learners, chemistry, and

artwork, and he has reviewed much o my writing

Ac kno wle dg me nts

vii

COAUTHORS AND CHAPTER REVIEWERS

I am deeply indebted to John T omas or his

contribu-tions, which involved designing this book and writing dra s

o several chapters: Clinical ests Based on DNA or RNA;

Basic Genetics or Biochemistry; ranscription and RNA

Processing; ranslation and Posttranslational Protein

Pro-cessing; Pentose Phosphate Pathway, Oxidative Stress, and

Glucose 6-Phosphate Dehydrogenase De ciency; Oxidative

Phosphorylation and Mitochondrial Diseases; Fatty Acids,

Ketone Bodies, and Ketoacidosis; riglycerides and

Hyper-triglyceridemia; Cholesterol Metabolism and

Hypercholester-olemia; Steroid Hormones and Vitamin D; Eicosanoids; and

Signaling

John T omas, PhD

Research Associate Pro essor (Retired)Department o Biochemistry and Molecular PharmacologyNew York University School o Medicine

New York, New York

I am very thank ul to Emine Ercikan Abali or coauthoring the chapters on riglycerides and Hypertriglyceridemia and Cholesterol Metabolism and Hypercholesterolemia

Emine Ercikan Abali, PhD

Associate Pro essor o Biochemistry and Molecular BiologyRutgers Robert Wood Johnson Medical School

Piscataway, New Jersey

I am very grate ul to the ollowing persons or contributing their expertise and reviewing chapters:

Philadelphia, Pennsylvania

James C Barton, MD

Medical DirectorSouthern Iron Disorders Center;

Clinical Pro essor o MedicineDepartment o Medicine

University o Alabama at BirminghamBirmingham, Alabama

Jef rey L Benovic, PhD

Pro essorDepartment o Biochemistry and Molecular BiologySidney Kimmel Medical College at T omas Jef erson University

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Bruno Calabretta, MD, PhD

Pro essor

Department o Cancer Biology

Sidney Kimmel Medical College at T omas Jef erson

University

Gino Cingolani, PhD

Pro essor

Department o Biochemistry and Molecular Biology

University

Joe Deweese, PhD

Associate Pro essor

Department o Pharmaceutical Sciences

Lipscomb University, College o Pharmacy and Health

Sciences

Nashville, ennessee

ina Bocker Edmonston, MD

Associate Pro essor

Department o Pathology and Laboratory Medicine

Cooper University Health Care, Cooper Medical School at

Associate Pro essor

Department o Molecular Physiology and Biophysics

University

Andrzej Fertala, PhD

Pro essor

Department o Orthopaedic Surgery

Steven K Herrine, MD

Pro essorDepartment o MedicineSidney Kimmel Medical College at T omas Jef erson University

Jacqueline M Hibbert, PhD

Associate Pro essorDepartment o Microbiology, Biochemistry and Immunology

Morehouse School o MedicineAtlanta, Georgia

Jan B Hoek, PhD

Pro essorDepartment o Pathology, Anatomy, and Cell BiologySidney Kimmel Medical College at T omas Jef erson University

anis Hogg, PhD

Associate Pro essorDepartment o Medical Education exas ech University Health Sciences Center El Paso, Paul

L Foster School o Medicine

El Paso, exas

Ya-Ming Hou, PhD

Serge A Jabbour, DM

Pro essorDepartment o MedicineSidney Kimmel Medical College at T omas Jef erson University

Francis E Jenney Jr, PhD

Associate Pro essorDepartment o Biomedical SciencesGeorgia Campus–PCOM

Suwanee, Georgia

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Clinical Associate Pro essor

Department o Medicine, Division o Gastroenterology and

University o Utah, School o Medicine

Salt Lake City, Utah

Diane Merry, PhD

Pro essor

Associate Pro essor

Department o Biochemistry and Molecular Medicine

University o Montreal

Montreal, Québec, Canada

Lawrence Prochaska, PhD

Pro essor

Wright State University, Boonsho School o Medicine

Dayton, Ohio

Prasad Puttur, PhD

Pro essor

Medical College o Georgia at Augusta University

Augusta, Georgia

Lucy C Robinson, PhD

Associate Pro essor

Louisiana State University Health Sciences Center

Shreveport, Louisiana

Lukas Ronner, BS

Medical StudentIcahn School o Medicine at Mount SinaiNew York, New York

John Sands, PhD

Pro essorDepartment o BiochemistryRoss University, School o MedicinePicard, Dominica

Charles Scott, PhD

Director, Jef erson Discovery CoreDepartment o Biochemistry and Molecular BiologySidney Kimmel Medical College at T omas Jef erson University

Philip Wedegaertner, PhD

Charlene Williams, PhD

Pro essorDepartment o Biomedical SciencesCooper Medical School o Rowan UniversityCamden, New Jersey

Edward Winter, PhD

akashi Yonetani, PhD

Pro essorDepartment o Biochemistry and BiophysicsPerelman School o Medicine o the University o Pennsylvania

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FRANK H NETTER, MD

Frank H Netter was born in 1906 in New York City He

studied art at the Art Student’s League and the National

Academy o Design be ore entering medical school at New

York University, where he received his MD degree in 1931

During his student years, Dr Netter’s notebook sketches

attracted the attention o the medical aculty and other

phy-sicians, allowing him to augment his income by illustrating

articles and textbooks He continued illustrating as a

side-line a er establishing a surgical practice in 1933, but he

ulti-mately opted to give up his practice in avor o a ull-time

commitment to art A er service in the United States Army

during World War II, Dr Netter began his long

collabora-tion with the CIBA Pharmaceutical Company (now

Novar-tis Pharmaceuticals) T is 45-year partnership resulted in

the production o the extraordinary collection o medical

art so amiliar to physicians and other medical pro essionals

worldwide

In 2005, Elsevier, Inc purchased the Netter Collection and

all publications rom Icon Learning Systems T ere are now

over 50 publications eaturing the art o Dr Netter available

through Elsevier, Inc (in the US: www.us.elsevierhealth.com/

Netter and outside the US: www.elsevierhealth.com)

Dr Netter’s works are among the nest examples o the use

o illustration in the teaching o medical concepts T e 13-book Netter Collection o Medical Illustrations, which includes the greater part o the more than 20,000 paintings created by Dr Netter, became and remains one o the most amous medical works ever published T e Netter Atlas o Human Anatomy, rst published in 1989, presents the anatomical paintings rom the Netter Collection Now translated into 16 languages, it is the anatomy atlas o choice among medical and health pro es-sions students the world over

T e Netter illustrations are appreciated not only or their aesthetic qualities, but, more important, or their intellectual content As Dr Netter wrote in 1949, “ clari cation o a subject is the aim and goal o illustration No matter how beauti ully painted, how delicately and subtly rendered a subject may be, it is o little value as a medical illustration i it does not serve to make clear some medical point.” Dr Netter’s planning, conception, point o view, and approach are what

in orm his paintings and what make them so intellectually valuable

Frank H Netter, MD, physician and artist, died in 1991.Learn more about the physician-artist whose work has inspired the Netter Re erence collection: http://www.netterimages.com/artist/netter.htm

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T is book provides an introduction to and review o

biochem-istry as it pertains to the competencies required or graduation

as a doctor o medicine or pharmacy Increasingly, the basic

sciences are taught alongside clinical science, o en organ by

organ T is book can help students in such integrated

curri-cula gain a discipline-speci c understanding o biochemistry,

particularly metabolism T e book is structured so that it is

use ul or both the novice and the student who needs a quick

review in preparation or licensure exams T e chapters are

extensively cross-re erenced so the material can be used in

almost any chapter sequence Descriptions o disease states are

a regular part o the book rather than an addendum in the

margin Students o en nd it challenging to use their

knowl-edge o basic science to solve clinical problems Hope ully, Dr

Netter’s images (“Medicine’s Michelangelo”), as well as the text

and other diagrams in this book, will help students build

mental bridges between basic science and clinical practice

T e chapters have a structure that makes it easy or the

reader to decide what to read and review:

■ T e Synopsis is an introductory overview o the content o

the chapter that requires very little preexisting knowledge

■ T e Learning Objectives indicate what the reader should

be able to do when mastering the material presented in the

chapter

■ Each section starts with a preview

■ Selected terms are printed in bold to make it easier to nd relevant text when starting rom the index

■ T e diagrams contain only the most essential in ormation

■ T e Summary provides a brie overview o the chapter material or the expert

■ A Further Reading section provides the reader with a ing point to satis y deeper interests

start-■ Review Questions provide the reader with an opportunity

to apply newly acquired knowledge Answers to these tions are at the end o the book

ques-Writing this text and designing the accompanying graphs has been a wonder ul and interesting journey or me I have also enjoyed many years o teaching biochemistry to uture physicians and pharmacists I hope that you, the reader, will also be amazed by the processes that underlie human exis-tence, both in health and in sickness

Peter Ronner

P.S.: Please eel ree to email suggestions or improvements to peterronner1@gmail.com

Pre fac e

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13 Pathologic Alterations of the Extracellular Matrix That Involve Fibrillin, Elas tin,

21 Pentos e Phos phate Pathway, Oxidative Stres s , and Glucos e 6-Phos phate Dehydrogenas e

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LEARNING OBJECTIVES

For mastery o this topic, you should be able to do the ollowing:

■ Describe the components and architecture of a DNA double

helix and explain where proteins bind to DNA helices.

■ Provide an example of reporting a DNA sequence in the

cus-tomarily abbreviated style.

■ Describe the most basic unit for packaging DNA into the nucleus.

■ Describe the normal human karyotype and list the number

of DNA double helices that make up a single metaphase

chromosome.

■ Describe the function of DNA topoisomerases and explain

the role of these enzymes in changing the topology of

chromosomes.

As part o epigenetic regulation, ~4% o the cytidine

nucleotides o DNA in the nucleus are methylated to

5-methyl deoxycytidine (see Fig 1.1) T e term epigenetic regulation re ers to changes in the DNA or DNA-associated proteins that do not af ect the sequence o the bases but af ect gene expression Some o these changes can be heritable and passed rom one cell to its descendants (see imprinting in Chapter 5) Quite generally, methylation in uences the higher-order packing and transcription o DNA (see Chapter

6) Methylation is required or the inactivation o the second

X chromosome in emales (see Chapters 5 and 21), the ing o certain transposons (movable genetic elements), regu-lation o the expression o genes during development, and determining the expression o particular genes rom only the mother or only the ather

silenc-Each DNA molecule has a 5′ end and a 3′ end (Fig 1.2)

o distinguish the atoms o the deoxyribose rom those o the

base, the deoxyribose carbon atoms are given a prime as a

post x (e.g., 3′ ) T e dinucleotide shown in Fig 1.2 has

a phosphate group at the 5′ position o nucleotide 1 and a hydroxyl group at the 3′ position o nucleotide 2, which is

typical o DNA T e nucleotides are linked by phosphodiester

bonds DNA is normally elongated at the 3′ end (see Section

1 in Chapter 3)

By convention, the sequence o a DNA is written as the

sequence o the bases in the 5′→3′ direction, using A or

adenine, C or cytosine, G or guanine, and or thymine I

the sequence is instead written 3′→5′, this must be indicated

T e sequence o bases in DNA contains heritable in ormation DNA is ound in the nucleus (see Section 4) and in mitochon-dria (see Section 3 in Chapter 23)

COMPLEMENTARY BASES

In the ashion o a zipper, complementary DNA molecules associate by hydrogen bonding A and can be linked by two hydrogen bonds, C and G by three hydrogen bonds

In Watson-Crick base pairing, A and are hydrogen bonded to each other, and so are C and G Each base o a nucleotide contains one or more hydrogen donors (–OH and –NH2) and one or more hydrogen acceptors (=O and =N–) A hydrogen acceptor can orm a partial bond to a donor’s hydro-

gen atom; such a bond is called a hydrogen bond A and

each contain one hydrogen donor and one hydrogen acceptor

in suitable positions, such that A and can be linked by a total

o two hydrogen bonds (Fig 1.3) C has one hydrogen donor and two hydrogen acceptors, while G has two hydrogen donors

Chapte r

1 Human Karyo type and the Struc ture o f DNA

SYNOPSIS

■ Heritable information is encoded in deoxyribonucleic acid (DNA)

DNA is a linear polymer of deoxyribonucleotides, and it is present

in the nucleus and mitochondria of cells.

■ The DNA of a cell comprises pairs of complementary molecules;

each pair assumes a double-helical structure.

■ DNA double helices in the nucleus are wound into higher-order

structures The simplest of such structures is the nucleosome;

the most complex structures exist in the form of condensed

chromosomes during cell division Light microscopic

examina-tion of these chromosomes is part of karyotyping.

■ Helicases and topoisomerases change the coiling of DNA for

transcription, replication, and repair of DNA.

■ Inhibitors of DNA topoisomerases can be used to destroy cancer

cells or bacteria.

Mitochondria and the nucleus o each cell contain DNA that

is a polymer o our basic types o nucleotides DNA stores

heritable in ormation by way o its nucleotide sequence

DNA is a linear polymer o the deoxyribonucleotides

deoxyadenosine monophosphate (dAMP), deoxyguanosine

monophosphate (dGMP), deoxycytidine monophosphate

(dCMP), and thymidine monophosphate (d MP, MP; Fig

1.1) Each deoxyribonucleotide consists o

deoxyribosephos-phate (derived rom a pentose, a 5-carbon sugar) covalently

linked to a base that is adenine, guanine, cytosine (or 5-methyl

cytosine), or thymine Adenine and guanine structurally

resemble purine; hence, they are called purine nucleotides

(synthesis, turnover, and degradation o these nucleotides are

described in Chapter 38) Cytosine and thymine structurally

resemble pyrimidine; hence, they are called pyrimidine

nucleotides (synthesis o these nucleotides is described in

Chapter 37)

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Fig 1.1 Struc ture s o f de o xyribo nuc le o tide s fo und in DNA. The as teris k indicates the s ite of potential cytos ine methylation

O OH

CH 2

N O

H

H H

O OH

H

H H

O OH

CH2

–

O O

O–P O

O OH

N N

xy-Bas e

De o ribo s e pho s phate

H H

die s te r

O O

O–P

H H

5´

3´

H

H H

O OH

CH 2 Bas e 2

and one hydrogen acceptor in suitable positions so that C and

G can be linked by a total o three hydrogen bonds Since they

orm hydrogen bonds with each other, A and are called

complementary bases; likewise, C and G are complementary

bases CG base pairs are harder to separate than A base pairs

because they have more hydrogen bonds (Non–Watson-Crick

base pairing is observed predominantly in RNA, where it is

common.)

In two complementary DNA molecules, all bases orm

hydrogen-bonded A and GC base pairs, and the molecules

are paired in an antiparallel ashion For instance, the

mole-cules 5′-AACG -3′ and 3′- GCA-5′ are complementary

(Fig 1.4) T e nucleotide at the 5′ end o one DNA strand is

thus hydrogen bonded to the nucleotide at the 3′ end o its

complementary DNA strand All heritable human DNA exists

in complementary strands that, in vivo, usually assume a double-helical structure (Fig 1.5) In mitochondria, each DNA strand consists o about 16,000 nucleotides; in the nucleus, each DNA strand consists o more than 45 million nucleotides

When a DNA sequence is reported, the sequence o the complementary strand is usually omitted because it can easily

be in erred

According to the Chargaf rule, DNA contains equimolar

amounts o A and , as well as equimolar amounts o C and G A and CG base pairing are the basis o Chargaf ’s nding

Most human DNA assumes a double-helical structure T e double helix consists o two complementary strands that run

in opposite directions

Complementary hydrogen-bonded DNA molecules

nor-mally assume the structure o a DNA double helix (see Fig.1.5) In this structure, the hydrogen-bonded bases are close to the central long axis o the DNA helix T e covalently linked deoxyribose phosphates o the two DNA strands wind around the periphery o the helix cylinder, akin to the threads o an unusual screw (a typical screw has only one thread) As is evident rom Fig 1.3, the bonds between the bases and the deoxyribose moieties (i.e., the N-glycosidic bonds) do not point in exactly opposite directions Hence, the two strands o linked deoxyribose phosphates are closer together on one side

o the base pair than on the other side T us, the DNA double

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Human Karyotype and the Structure of DNA 3

In vivo, hydrogen bonds between bases o complementary DNA strands are broken and re ormed during replication, repair, or transcription o DNA (see Chapters 2, 3, and 6) In vitro, the separation and “rejoining” (hybridization) o com-plementary DNA strands are an important part o many diag-nostic DNA-based procedures (see Chapter 4)

INTO CHROMATIDS

T e length o human DNA molecules ar exceeds the diameter

o the cell nucleus DNA is compacted into orderly structures ranging rom nucleosomes to metaphase chromatids

In the nucleus, DNA is olded into nucleosomes, which in turn are part o increasingly higher orders o olding T e greatest degree o DNA compaction is needed or cell division

T e longest human chromosome (chromosome 1) contains about 246 million base pairs and has a length ~15,000 times the diameter o a typical nucleus T e organization o DNA also af ects the transcription o genes T e basic unit o olding

is the nucleosome, o which several types exist Nucleosomes contain a core particle that consists o eight histone proteins,

a DNA helix o ~147 base pairs that encircles the histones ~1.7

times (Fig 1.6), and linker DNA o ~40 base pairs to which histone H1 is o en bound N- and C-terminal tails o the

histones protrude rom nucleosome core particles Certain amino acids in these histone tails can be modi ed ( able 1.1)

T e resulting structure o the histone tails af ects the packing,

replication (see Chapter 3), and transcription o DNA (see

Chapter 6)

Nucleosomes can be organized into 30-nm diameter

chromatin bers Chromatin bers, in turn, can be

con-densed into yet higher-order structures, and nally into

helix has unequal grooves: a minor groove and a major

groove.

T ere are several double-helical DNA structures that dif er

in handedness, diameter, and rise per turn T e most

promi-nent o these structures are re erred to as A-DNA, B-DNA,

and Z-DNA In cells, most DNA is in a double-helical orm

that resembles B-DNA

ranscription actors that bind to DNA (see Chapter 6)

bind to atoms at the sur ace o the major or minor groove and

can thereby recognize a particular nucleotide sequence Some

transcription actors increase the contact with DNA urther

by bending or partially opening the double helix

Certain positively charged side chains o DNA-binding

proteins, as well as certain positively charged stains used in

histochemistry (e.g., the basic dyes hematoxylin, methylene

blue, and toluidine blue), bind to DNA by interacting

with the negatively charged phosphate groups T ese

phos-phate groups line the backbone o DNA and are exposed

on the outside o the double helix (see Fig 1.5) Among

DNA-binding proteins, positive charges are ound on some

amino acid side chains o histones (see below) and o certain

transcription actors (see Chapter 6) Complexes o DNA

and the DNA-binding histone proteins are re erred to as

chromatin T e negative charges o the phosphate groups o

DNA alone give rise to an overall negative charge o DNA

that is taken advantage o in the electrophoresis o DNA

(see Chapter 4)

Fig 1.3 Hydro g e n bo nding be twe e n c o mple me ntary bas e s

O O–

O–P O

–

O O

O–P O

N N

CH2

O O–

O–P

O

H2C O

O

N-glycos idic bond

dCMP

dGMP

O

Fig 1.4 Bas ic s truc ture o f do uble -s trande d DNA.

Double-s tranded DNA can form a double helix (Double-s ee Fig 1.5 )

G C

C

A

A A

5´

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chromatids Chromatids are ound only in dividing cells

during mitosis

T e cellular processes o DNA repair, replication, and scription (discussed in Chapters 2, 3, and 6) require, at times, the unwinding o DNA rom its structures (e.g., the 30-nm chromatin f ber, the nucleosomes, and the double helix) ol-lowed by rewinding Changes in the winding o DNA are catalyzed by helicases and topoisomerases

opology is a eld o mathematics that describes the de mation, twisting, and stretching o objects such as DNA As outlined above, DNA o human chromosomes is organized into nucleosomes and higher-order structures T e chemical structure o DNA can accommodate only a limited amount o torsional strain, and the chromatin structure prevents the dis-persion o strain over a large distance (As an analogy, con-sider how winding af ects the three-dimensional shape o a phone cord or garden hose.) T us, the winding o DNA (the topological state o DNA) matters orsional strain can result rom the partial opening o a DNA helix (e.g., during repair, replication, or transcription) or rom a nonrotatable complex

or-o enzymes that mor-oves in between the twor-o strands or-o a DNA double helix (Fig 1.7) Replication and transcription, or example, cause overwinding, or positive supercoiling, within the chromosomes

Helicases can use energy rom A P hydrolysis to separate

the two strands o the double helix T e energy input rom

A P is needed to pay the penalty or breaking hydrogen bonds between bases in DNA Humans produce several dif erent helicases T e physiological roles o these helicases are largely unknown Mutations in a ew helicases are known to cause

disease: a de ciency in WRN causes Werner syndrome

(predominantly characterized by premature aging); a de

-ciency in BLM causes Bloom syndrome (accompanied by an

increased rate o tumorigenesis); and a de ciency in RECQ4

causes Rothmund-T omson syndrome (associated with skin

Comple me nta ry DNA stra nds

De ribos e

oxy-65°

H-bonde d

ba s e pa ir

Majo r g ro o ve

Mos t DNA binding

prote ins re a d the

nucle otide s e que nce

Fig 1.5 The do uble -he lic al s truc ture of DNA. The s tructure of

an 11-bas e-pair s egment of the human N-ras gene is s hown (the

s equence of the purple s trand is 5′-GGCAGGTGGTG; this s equence

frequently undergoes mutation and then promotes the development of

a tumor) The bas es are in the center, and the ribos es are located in the periphery of the helix The blue and purple s naking cylinders are imagi- nary forms that connect the phos phorus atoms and s how the progres s

of the helix The bonds that connect phos phates and ribos es and that form the true backbone of a DNA s trand are generally s ituated jus t outs ide the calculated cylinders The planes of the rings of deoxyribos es and bas es are s hown in light gray The two DNA s trands are antiparallel;

the blue s trand is winding downward (5′ to 3′ ), while the purple s trand

is winding its way up (5′ to 3′ ) The s tructure of this oligomer mos tly

res embles the s tructure of B-DNA (Bas ed on Protein Data Bank [ www

oligodeoxynucleotide containing the human N-ras codon 12 s equence

re ned from 1H NMR us ing molecular dynamics res trained by nuclear

Overhaus er effects Chem Res Toxicol 1996;9:114–125.)

Trang 21

Fig 1.6 Pac king of DNA into a nuc le os ome c ore partic le in the nuc le us Nucleotides are

s hown in black An idealized cylinder through all phos phorus atoms is s hown in light brown DNA winds almos t twice around a core of eight his tone proteins There are two copies each of his tone H2A (gold), H2B (red), H3 (blue), and H4 (green) (Bas ed on Protein Data Bank [ www.rcs b.org ] le 1KX5 from Davey CA, Sargent DF, Luger K, Maeder AW, Richmond TJ Solvent mediated interactions in the s tructure of the

nucleos ome core particle at 1.9 Å res olution J Mol Biol 2002;319:1097–1113.)

90°

DNA His tone s

His tone tails

DNA 5´

S e e n witho ut his to ne s : 5´

Table 1.1 Mo di c ations o f His to ne s

Acetylation (CH3–CO– is an acetyl group) Ubiquitylation (ubiquitin is a 76-residue protein) Sumoylation (SUMO = small ubiquitin-like modi er, a small group of ~100-residue proteins) ADP-ribosylation (conjugation with a ribose that in turn forms a phosphodiester with ADP)

Deimination (exchange of =NH for =O, turning arginine into citrulline)

Trang 22

Fig 1.7 Strain impo s e d o n do uble -he lic al DNA whe n the

he lix is ope ne d up partially, o r whe n a no nro tatable o bje c t

mo ve s in be twe e n the two c o mple me ntary s trands

Fig 1.8 To po is o me ras e I c uts o ne s trand o f DNA and s wive ls aro und the othe r s trand. The ribos es and bas es of DNA are s hown

in black An arti cial, s moothed backbone is drawn through the phos phorus atoms in red, brown, or orange (there are three s egments of DNA) The enzyme is s hown in greenis h blue A tyros ine res idue (magenta)

-is covalently linked to the 3′ end of the “red” DNA chain The “brown”

DNA chain, together with a portion of the “orange” DNA chain, can rotate

and thereby relieve tors ion s tres s Normally, the 5′ end of the “brown” chain then reconnects with the 3′ end of the “red” chain Here, the che-

motherapeutic drug topotecan (s hown as a s tick model with C in grey,

N in blue, and O in red) binds in between the 3′ bas e of the “red” chain and the 5′ bas e of the “brown” chain; topotecan thereby prevents religa-

tion of thes e chains , which leads to cell death (Bas ed on Protein Data Bank [ www.rcs b.org ] le 1K4T from Staker BL, Hjerrild K, Fees e MD, Behnke CA, Burgin J r AB, Stewart L The mechanis m of topois omeras e

I pois oning by a camptothecin analog Proc Natl Acad S ci 2002;

DNA (double he lix)

abnormalities) All o these disorders are rare and show

auto-somal recessive inheritance

Once a part o two complementary DNA strands has been

separated, single-strand binding proteins (e.g., replication

protein A [RPA]) can prevent the pairing o bases

opoisomerases can relieve strain in DNA and thus alter

the topology o DNA Supercoiled DNA is DNA that has

olded back on itsel to accommodate under- or overwinding

(negative or positive supercoiling, respectively) o the double

helix opoisomerase I and topoisomerase II both relax

super-coiled DNA during replication and transcription

opoisom-erase II also untangles (decatenates) DNA or chromosome

segregation during mitosis ype I topoisomerases cut one

strand, whereas type II topoisomerases cut both strands o a

double helix In both cases, the enzyme orms a transient

covalent link with either the 5′ or 3′ end o the broken DNA.

ype I topoisomerases (including topoisomerase I) relieve

the torsional strain o DNA by cutting one strand o the double

helix, swiveling around the intact strand or passing the intact

strand through the break, and then ligating the cut strand

again (Fig 1.8)

Inhibitors o topoisomerase I of er a means o pre erentially

damaging tumor cells that divide more requently than normal

cells Analogs o camptothecin prolong the li etime o a

cova-lent DNA–topoisomerase I complex that is ormed as a normal

reaction intermediate As the genome is copied during

replica-tion (see Chapter 3), the obstructing DNA–topoisomerase I–

camptothecin complex can result in permanent strand breaks,

which the cell may attempt to repair When the number o

double-strand breaks exceeds a cell’s capacity or repair (see

homologous recombination repair in Chapter 2), the cell

undergoes apoptosis (i.e., programmed cell death; see Chap

-ters 2 and 8) Camptothecin analogs (e.g., topotecan and

iri-notecan) are used predominantly in the treatment o advanced

malignancies (e.g., relapsing small-cell lung cancer or

meta-static ovarian cancer)

ype II topoisomerases (topoisomerase II in humans, and

DNA gyrase and topoisomerase IV in bacteria) cleave both

strands o one double helix, use con ormational changes in the enzyme subunits to pass a separate DNA segment between the break, and then ligate the cut strands (Figs 1.9 and 1.10) T is process requires A P ype II topoisomerases are involved in relaxing the supercoils that result rom DNA replication or

Trang 23

increase a patient’s risk o developing therapy-related mia Aside rom poisons, catalytic inhibitors o topoisomerase

leuke-II inhibit other portions o the catalytic mechanism o the enzyme (e.g., A P hydrolysis) and cause cell death without inducing DNA strand breaks

Some polyphenols in our diet also poison topoisomerase

II Soybeans contain genistein, which binds to estrogen

recep-tors and can help ameliorate symptoms o menopause tein also poisons topoisomerase II Genistein appears to have anticancer activity, but in pregnant mothers it also con ers a higher risk o childhood leukemia in the of spring Green tea

Genis-contains the polyphenol epigallocatechin gallate (EGCG),

which also poisons topoisomerase II T e biological impact o these poisons has not been ully established, and there is some evidence that these agents may be chemopreventive

Fluoroquinolone antibacterials inhibit bacterial DNA

gyrase (the name given to the positively supercoiling somerase II in bacteria) and topoisomerase IV Commonly

topoi-used quinolones are the broad-spectrum antibiotics

oxacin, levo oxacin, o oxacin, and moxi oxacin.

transcription Sister chromatids become intertwined during

DNA replication; this linking is called catenation T e

essen-tial unction o type II topoisomerases, which cannot be

per ormed by type I enzymes, is the separation (decatenation)

o replicated chromosomes be ore compaction and cell

division

Inhibitors o topoisomerase II are use ul as anticancer

agents Most o these inhibitors are part o a class called

topo-isomerase II poisons wo drugs o this class that are widely

used in chemotherapy are doxorubicin (an anthracycline) and

etoposide (an epipodophyllotoxin; Fig 1.11) In the presence

o these drugs, topoisomerase II can cleave DNA but cannot

ligate it T ere ore, DNA replication and transcription are

both inhibited As a consequence, DNA strand breaks

accu-mulate and lead to apoptosis (programmed cell death)

However, these drugs are also mildly mutagenic and thus

Fig 1.10 Human to po is o me ras e II α c atalyze s the pas s ag e

o f o ne DNA s trand thro ug h ano the r DNA s trand. The enzyme

functions as a dimer The image s hows the catalytic core domain,

includ-ing the central gate and the lower, C-terminal gate; not shown is the

ATPase domain, which is at the top of the s tructure (Based on Protein

Data Bank [ www.rcs b.org ] le 4FM9 from Wendorff TJ , Schmidt BH,

Heslop P, Austin CA, Berger J M The structure of DNA-bound human

topois omeras e II alpha: conformational mechanis ms for coordinating

inter-s ubunit interactions with DNA cleavage J Mol Biol 2012;424:

3 This ga te (inte rfa ce

be twe e n two

s ubunits ) ope ns

2 This ga te clos e s

Fig 1.11 Eto po s ide is an inhibito r o f to po is o me ras e II and

is o fte n us e d in the tre atme nt o f e xte ns ive s mall-c e ll lung

c anc e r. Thes e patients typically have dis s eminated dis eas e Etopos ide

is often combined with a platinum drug

Trang 24

Chapter 3) T en, each one o these helices is greatly

con-densed into chromatids (see Section 4) Proteins join pairs

o identical chromatids at their centromeres to orm

meta-phase chromosomes.

With basophilic stains (e.g., Giemsa stain), metaphase

chromosomes can be visualized under a light microscope (Fig

1.12) Images o stained chromosomes are used to characterize

the chromosomes o an individual (i.e., to describe an

indi-vidual’s karyotype) Stains used in karyotyping produce

various diagnostically use ul banding patterns, which depend

on the staining procedure used, the degree o DNA

compac-tion, and the presence o DNA-bound proteins

wo o the 46 chromosomes are called sex chromosomes;

the remaining 44 chromosomes are called autosomes

Humans typically inherit one sex chromosome and 22

auto-somes rom each parent T ere are two types o sex

chromo-somes, X and Y Each emale with a normal karyotype has two

X chromosomes (one o which gets inactivated by

methyla-tion; see Chapter 5) Each male with a normal karyotype has

one X and one Y chromosome T e 22 autosomes are

num-bered rom 1 to 22 in approximate order o decreasing size

(see Fig 1.12)

Segregation o chromosomes occurs during both cell

divi-sion (mitosis) and gamete ormation (meiosis) During

mitosis, pairs o chromatids are pulled apart so that each

daughter cell gets 46 chromatids (i.e., 46 DNA double helices)

In nondividing cells, the term chromosome is used to

desig-nate a single chromatid (i.e., a single DNA double helix) Every

cell in the G0 phase o the cell cycle has 46 chromosomes

(in this case, 46 DNA double helices) During meiosis I,

Fig 1.12 No rmal male karyo g ram. For karyotyping, cultured cells

are arres ted in metaphas e This karyogram s hows the light-micros copic

images of s tained chromos omes from a s ingle cell The chromos omes

are s orted and analyzed according to their s ize and banding pattern

(Courtes y Dr Barry L Barnos ki, Oncocytogenetics Laboratory, Cooper

Univers ity Hos pital, Camden, NJ )

meiosis II, paired chromatids are pulled apart to yield cells

that contain only 23 chromatids (i.e., 23 DNA double helices).Cells contain more than 100 times more DNA in their nucleus than in their mitochondria Although a cell’s network

o mitochondria contains thousands o copies o

mitochon-drial DNA, even the shortest o the 46 chromosomes contain

more than 3000 times the number o base pairs in the chondrial genome

pair in a head-to-tail ashion (i.e., the 5′ end o one strand

is paired with the 3′ end o its complementary strand)

Unless indicated otherwise, DNA sequences are written in

a 5′→3′ direction.

■ DNA binding proteins can bind selectively to a speci c DNA sequence by interacting with the atoms o bases that are at the sur ace o the DNA helix grooves

■ T e length o nuclear DNA molecules ar exceeds the eter o the nucleus Inside the nucleus, most o the DNA is condensed into nucleosomes; this, in turn, is condensed into higher-order structures T ese structures play critical roles in the regulation o transcription and make the orderly separation o DNA molecules possible during cell division

diam-■ Helicases separate complementary strands o DNA stranded DNA binding proteins prevent the pairing o separated strands opoisomerases cut one or both strands

Single-o dSingle-ouble-helical DNA, relieve tSingle-orsiSingle-onal strain (tSingle-opSingle-oisSingle-om-erases I and II) or untangle chromosomes in preparation

or mitosis (topoisomerase II), and then religate the strands Inhibitors o topoisomerases are used in chemotherapy or cancer

■ Human cells with a normal karyotype contain 46 somes: 23 rom the mother and 23 rom the ather O the chromosomes, 44 are autosomes and two are sex chromosomes

chromo-FURTHER READING

■ Deweese JE, Osherof MA, Osherof N DNA opology and topoisomerases: teaching a “knotty” subject Biochem Mol Biol Educ 2008;37:2-10

■ Ozer G, Luque A, Schlick T e chromatin ber: multiscale problems and approaches Curr Opin Struct Biol 2015;31:124-139

Trang 25

Re vie w Que s tio ns

? ?

■ essarz P, Kouzarides Histone core modi cations

regu-lating nucleosome structure and dynamics Nat Rev Mol

Cell Biol 2014;15:703-708

■ Vos SM, retter EM, Schmidt BH, Berger JM All tangled

up: how cells direct, manage and exploit topoisomerase

unction Nat Rev Mol Cell Biol 2011;12:827-841

■ Wapner RJ, Martin CL, Levy B, et al Chromosomal

micro-array versus karyotyping or prenatal diagnosis N Engl J

Med 2012;367:2175-2184

■ Zhu P, Li G Structural insights o the nucleosome and the

30-nm chromatin ber Curr Opin Struct Biol 2016;

36:106-115

1 T e gure above shows part o a nucleosome T e three

pentagons identi ed by arrows represent which o the

A Modi cation o histone tails

B Pairing o complementary bases

C Reading o bases in the major groove

D Relaxation o supercoiled DNA

4 Many DNA-based diagnostic tests use a DNA polymerase rom T ermus aquaticus, a bacterium that can survive high temperatures Compared with the DNA o bacteria that grow at 25°C, the DNA o aquaticus is expected to have

a higher raction o which o the ollowing nucleotides?

Trang 26

SYNOPSIS

■ DNA damage may be due to the inherent properties of DNA or

the damaging effects of ultraviolet light, radiation, drugs, or

noxious agents in the environment Damage may manifest as

lesions to nucleotides, DNA adducts, crosslinks within or

between DNA strands, or single- or double-strand breaks in the

DNA Diverse DNA repair mechanisms exist, ensuring near

con-stancy of the genome.

■ Knowledge of DNA repair is important for understanding how

inadequate DNA repair leads to tumorigenesis and how

chemo-therapy and radiochemo-therapy of cancer can lead to overwhelming

damage and death of tumor cells, as well as neoplasms among

previously normal cells.

■ DNA repair has been studied extensively in bacteria and yeast

Although humans have more complex DNA repair pathways

than these single-cell organisms, DNA repair proteins are highly

evolutionarily conserved Appropriately, many human DNA repair

proteins are named after their counterparts in bacteria and

yeast.

■ The base-excision repair pathway becomes active when a single

nucleotide is altered The faulty nucleotide is excised and

replaced with a new one that ts the complementary DNA

strand.

■ The mismatch repair pathway detects mismatches of base pairs

and bulges due to missing or excess nucleotides that arise from

faulty DNA replication The most recently synthesized portion of

DNA is removed, and new DNA is synthesized based on the

complementary DNA strand.

■ The nucleotide-excision repair pathway repairs damage that

grossly distorts DNA Such damage may stem from exposure

to the sun, cigarette smoke, or platinum chemotherapeutic

drugs A section of the damaged strand is cut out, and the DNA

is then resynthesized.

■ Nonhomologous end-joining repairs double-strand breaks by

joining the broken ends The product is often different from the

original DNA Double-strand breaks can arise from DNA damage

by x-rays or chemotherapeutic drugs.

■ The homologous recombination pathway repairs double-strand

breaks by producing long, single-strand overhangs that invade

a homologous DNA strand The invaded strand then serves as

a template for resynthesis of the lost DNA.

■ Hereditary and acquired defects in DNA repair favor

tumorigen-esis (e.g., in the colon, breast, ovaries, pancreas, and skin).

T e base-excision repair (BER) pathway deals with common orms o damage to a single nucleotide It removes an altered nucleotide, adds the proper deoxyribonucleotide, and seals the cut in the DNA About 1% o all patients who have colon

cancer have two de ective copies o the MU YH gene that

encodes an enzyme needed or BER

Every day, in every cell, thousands o bases in DNA are altered (Fig 2.1) Bases (mostly adenine or guanine) are spon-

taneously lost rom the DNA deoxyribose backbone Bases can be deaminated, especially 5-methylcytosine and cytosine,

which thus give rise to thymine and uracil, respectively

Hydroxyl radicals can react with bases; this happens

espe-cially with guanine, orming 8-oxo-guanine (also called

8-hydroxyguanine) T e physiological methyl-group donor

S-adenosylmethionine can react with adenine to orm

3-methyladenine Ionizing radiation (e.g., x-rays, γ-rays)

can ionize water (thereby giving rise to a hydroxyl radical), oxidize a base, cleave a base rom the deoxyribosephosphate backbone, or ragment a deoxyribose and thereby cut one o the complementary DNA strands

In the short-patch BER pathway, enzymes recognize a

damaged nucleotide and then replace it (Fig 2.2) Humans

produce many DNA glycosylases that slide along DNA,

rec-ognize deaminated, hydroxylated, or methylated bases, and remove them T is generates a substrate that is recognized by

AP endonuclease 1, which cuts the DNA where the base is missing Polynucleotide kinase/phosphatase (PNKP) then

phosphorylates the ree 5′ end and dephosphorylates the

adja-cent 3′ end Poly-ADP-ribose polymerase (PARP) binds to

the strand break and recruits the protein XRCC1, which

serves as a plat orm or recruiting other repair proteins T en,

DNA polymerase β excises the abasic deoxyribose and

replaces it with a proper new nucleotide Finally, DNA ligase

■ Summarize the major DNA repair pathways.

■ Describe how ultraviolet light and high-energy x-rays damage

DNA, and how this damage is repaired.

■ Describe how polycyclic aromatic hydrocarbons in cigarette

smoke damage DNA and how this damage is repaired.

■ Explain how platinum drugs and nitrogen mustards (e.g., phosphamide) damage DNA and how this damage is repaired.

cyclo-■ Describe and explain commonly used lab tests for DNA match repair in biopsy tissues.

mis-■ Explain the term microsatellite instability, describe a lab test for microsatellite instability, and link microsatellite instability to a

de ciency in a DNA repair pathway.

■ List hereditary cancer syndromes and specify the associated defects in DNA repair, as well as the pattern of inheritance List any modi cations in chemotherapy or radiotherapy of tumors that must be made for affected patients.

■ Describe how chemotherapy and radiotherapy kill tumor cells and how these treatments can be tumorigenic in normal cells.

Trang 27

DNA Repair and Therapy of Cancer 11

damaged one, the mismatch repair (MMR) pathway (see

Section 2) o en detects the error For instance, it recognizes

an A opposite a U ( rom deamination o C) or opposite an oxo-G ( rom the hydroxylation o guanine)

T e enzyme DNA MYH glycosylase, encoded by the

MU YH gene, partners with the MMR pathway (see Section

2) to excise A opposite 8-oxo-guanine (see Fig 2.1) T e BER pathway then replaces the 8-oxo-G with G MU YH stands

or MutY homolog ( rom bacteria)

About 1% o all patients who have colorectal cancer have

MU YH-associated polyposis (MAP), a disease that is

caused by de cient DNA MYH glycosylase activity T e

disease shows autosomal recessive inheritance In patients with MAP, G→ mutations accumulate (persistent A opposite 8-oxo-G yields a in place o 8-oxo-G in the next round o replication) Interestingly, such G→ mutations are ound in the same genes that are mutated or no longer transcribed

in some patients who have sporadic colorectal cancer (i.e., in patients who do not have MAP) In patients with MAP, colon cancer typically occurs in the late orties At this time, the colon o en contains tens to hundreds o polyps

PARP inhibitors are in clinical trials or patients who have

tumors with de ective homologous recombination (HR) repair (see Section 4) and there ore rely unusually heavily on PARP-dependent BER and nonhomologous end joining (NHEJ; see Section 4) PARP inhibitors inhibit BER and NHEJ because poly-ADP-ribose recruits DNA repair proteins (e.g., XRCC1)

to damaged sites in the DNA While PARP inhibitors are tively innocuous to normal cells, they are especially toxic to tumor cells that are de cient in BRCA1 or BRCA2, proteins that play a role in HR repair

T e mismatch repair (MMR) system handles improper base matches, as well as single-strand loops that stem rom inser-tions or deletions during replication Repair is directed to the most recently synthesized DNA strand MMR is de ective in

~10% to 20% o sporadic cancers o the colon, rectum, stomach, or endometrium Hereditary mutations that a ect MMR are the cause o Lynch syndrome, which most o en causes colorectal or endometrial cancer

T e MMR pathway detects noncomplementary base pairs and repairs them Mismatches may stem rom the ollowing (Fig 2.3): (1) spontaneous tautomerization o bases during DNA polymerization; (2) deamination o cytosine to uracil or

o 5-methylcytosine to thymine; (3) DNA polymerase error o inserting a base that is not complementary to the DNA strand that is being copied; and (4) DNA polymerase slippage in nucleotide sequence repeats

In MMR, MSH proteins recognize the damage and MLH

proteins help initiate the excision o a stretch o the most recently synthesized DNA (Fig 2.4) Heterodimeric MSH pro-teins (homologs o bacterial MutS proteins; able 2.1) detect mispaired and unpaired bases MLH proteins (homologs o bacterial MutL proteins) then attach to the MSH proteins In

Fig 2.1 Spo ntane o us alte rations o f DNA that are re paire d

by the bas e -e xc is io n re pair pathway.

O O

N

N H H

H N N

3

N N

NH2N

O O

NH2

NH N

CH2

H

H H

N H

O O

N H H

H

H O

N N

8-Oxo-7,8-dihydroG

N N O

IIIα seals the nick in the DNA strand to reestablish a

contigu-ous DNA molecule

A single de ect also triggers the long-patch BER pathway,

which replaces 2 to 10 consecutive nucleotides Certain

oxida-tion products o a deoxyribose must be removed via the

long-patch pathway In addition, this pathway completes some o

the repairs that cannot be completed by single-patch BER

I damage to a base is not repaired, DNA replication (see

Chapter 3) may insert an incorrect nucleotide, or it may come

to a halt until the damaged base is repaired Replication stops

when methyladenine (see Fig 2.1) is present so that BER can

replace methyladenine with adenine When a base is missing,

translesion DNA synthesis inserts a nucleotide into the newly

synthesized DNA strand, but this nucleotide may be the wrong

one I replication inserts an inappropriate base opposite a

Trang 28

the DNA strand, using the complementary strand as a

tem-plate Finally, DNA ligase I ligates the pieces o DNA.

Inactivation o the DNA MMR system leads to an increased

susceptibility to cancer, especially cancer o the colon,

stomach, or endometrium Cells with impaired MMR mulate mutations at a vastly increased pace T is leads to rameshi mutations that impair the production and unction

accu-o tumaccu-or suppressaccu-ors, prevent praccu-ogrammed cell death, accu-or alter signaling, transcription, or immune surveillance

T e tumors rom ~15% o patients who have colon cancer and ~20% each o patients who have endometrial or gastric

cancer have de ective MMR systems In patients with a radic orm o this cancer, the MMR de ciency is usually due

spo-to the methylation o the promoter o both copies o the

MLH1 gene T e methylation essentially abolishes the

expres-sion o the MLH1 protein

addition, MLH proteins bind to an exonuclease ethered to

MSH and MLH proteins, the exonuclease 1 (Exo1) begins the

degradation o the most recently synthesized DNA strand at

a nearby cut in the DNA (it is unclear how this cut arises)

DNA polymerase δ then resynthesizes the missing portion o

Fig 2.2 The s ho rt-patc h bas e -e xc is io n re pair pathway.

O P O

O

–

O O O P O

O Bas e (A, C, G, or T)

O P

O

Ne w bas e (A, C, G, or T)

O P O

O–P

OH

–

O O O P O

O Ba s e (A, C, G, or T)

O P

O

–

O O O P O

O Base (A, C, G, or T)

O P

P NKP DNA polyme ra s e

DNA liga s e

Fig 2.3 Caus e s o f bas e mis matc he s that are re paire d by the

mis matc h re pair pathway. Blue arrows indicate hydrogen bonding

for bas e pairing

Tauto me rizatio n c hang e s bas e pairing

De amination c hang e s bas e pairing

Mis inc o rporatio n during re plic atio n

Slippage during re plic ation

NH NH

Fig 2.4 DNA mis matc h re pair. Shown is the repair of a G–T mis match.

Mis ma tch due to uncorre cte d e rror of DNA polyme ra s e

G T

A T T

Mis s ing 3´–5´ phos phodie s te r bond (s ource is uncle a r)

Single nucle otide s

Nucle otide s

Mos t re ce ntly s ynthes ize d s tra nd

MS H prote ins de te ct mis ma tch MLH prote ins re cruit e xonucle a s e 1

DNA polyme ra s e , liga s e I

Trang 29

particular MMR protein, exhibit an absence or greatly reduced immunoreactivity or that protein o complicate matters (see able 2.1), a lack o MLH1 o en leads to the degradation o the PMS2 protein, and a lack o MSH2 leads to the loss o the MSH6 protein (but not vice versa) Results o immunohisto-chemical assays can be used or guidance in DNA-based testing or mutations, hypermethylation, and microsatellite instability (see below)

De ective MMR can be detected as the microsatellite

instability (MSI) o DNA Mi-crosatellites are 5- to 100- old

repeats o sequences that contain one to ve nucleotides [e.g., (A)16 or (G )9] Microsatellites are also called short tandem

repeats When a patient is tested or MSI, DNA is obtained

rom the excised tumor and occasionally also rom peripheral blood lymphocytes (the DNA in lymphocytes is assumed to

be representative o the DNA in the germline) By using PCR (see Chapter 4), DNA that contains certain microsatellites (e.g., the mononucleotide repeats BA 25 and BA 26 and the dinucleotide repeats D2S123, D5S346, and D17S250) is ampli-

ed and analyzed or size BA 26 is within the MSH2 gene, but the remaining microsatellites are outside the genes that encode MMR proteins In MMR-de cient tumor cells, these repeats usually become shortened, giving rise to a shorter piece

o PCR-ampli ed DNA Most tumors that have MSI show abnormal lengths o our or ve o the ve microsatellite

sequences mentioned A tumor is usually said to have high

MSI instability i two or more o the ve tested microsatellites

Table 2.1 Prote ins That Are Involve d in DNA Mis

-matc h Re pair in the Nuc le us

MutSβ

MSH6 † (for mismatches and ≤2 extra or missing nucleotides in microsatellites)

MutSα

INITIATION OF NUCLEOTIDE EXCISION

PMS2 † (used in most mismatch

*One partner can partially make up for another.

† Most patients with Lynch syndrome inherited an inactivating mutation in

Lynch syndrome is a hereditary predisposition to cancer

that is due to a germline mutation in a DNA MMR gene (Fig

2.5) At least 1 in 1,000 individuals has Lynch syndrome, and

about 3% o all patients who have colon cancer have Lynch

syndrome Most patients with Lynch syndrome inherited a

mutation that inactivates MSH2, MSH6, MLH1, or PMS2 (see

able 2.1) T e second copy o the gene or its associated

pro-moter then undergoes mutation or epigenetic inactivation (via

DNA methylation) in certain cells o the body, such that no

unctional protein is produced in these cells (e.g., in the colon)

Patients who have Lynch syndrome have a ~70% li etime risk

or cancer o the colon, ~40% risk or cancer o the

endome-trium, and ~15% risk or cancer o the stomach or an ovary

T ese cancers occur at an unusually early age (e.g., colon

cancer typically in the mid-40s) In contrast to patients who

have a sporadic tumor with de ective MMR mechanisms,

patients who have Lynch syndrome also have a mutant MMR

gene in blood lymphocytes Since only one de ective allele

needs to be inherited, the disease shows autosomal dominant

inheritance (see Chapter 5) T is means that i only one parent

is af ected, each of spring has a 50% chance o inheriting the

disease

Immunohistochemical detection o MMR proteins in a

tumor o the colon or endometrium is o en part o the

diag-nosis o an MMR de ciency T e tissue is commonly stained

or MLH1, MSH2, PMS2, and MSH6 Patients who have

spo-radic colon cancer or endometrial cancer due to

hypermeth-ylation o the promoter or the MLH1 gene do not show

immunoreactivity or MLH1 Similarly, patients who have

Lynch syndrome, and there ore only a mutant version o a

Trang 30

Once the damage is detected, helicases in FIIH (a protein

complex with roles in both GG-NER and C-NER) unwind nearby DNA and veri y the presence o damage (Fig 2.10) RPA binds to single-stranded DNA and prevents the re orma-

tion o hydrogen bonds between base pairs T e ERCC1-XPF

endonuclease complex cuts unwound DNA 5′ o the lesion A

section o ~30 nucleotides is removed and a DNA polymerase

(e.g., δ, ε, or κ) resynthesizes the missing region Finally, a

DNA ligase (I or III) links the 3′ end o the newly synthesized

region to the rest o the DNA strand

Patients with de cient C-NER mostly show impaired development, premature aging, and neurodegeneration; some also have increased sensitivity to UV light I many transcrip-tion sites are halted and stopped or long periods, the cell

undergoes programmed cell death (apoptosis; see Chapter 8).Patients who have abnormalities speci cally in GG-NER tend to have very early-onset cancer (in part induced by UV light) because error-prone translesion DNA polymerases (see Chapter 3) bypass the many unrepaired lesions during DNA replication

T e inadequate repair o intrastrand crosslinks promotes

the ormation o tumors Inadequate repair o UV-induced DNA damage plays a role in the development o basal cell

show a change in length I only one o the ve microsatellites

is unstable, there is low MSI I all ve microsatellites are

stable, the tumor is said to be microsatellite stable.

In patients who have tumors that show MSI, pathogenic

changes can occur in the lengths o the A26 microsatellite

BA 26 in the MSH2 gene, in the C8 microsatellite o the

MSH6 gene, in the A10 microsatellite o the tumor growth

actor β receptor 2 gene, or in the G8 tract o the gene or the

cell-death-inducing protein BAX Changes in the repeat

lengths o the other our diagnostically measured

microsatel-lites (BA 25, D2S123, D5S346, D17S250) are not known to be

pathogenic

Patients who have a colon tumor that demonstrates

micro-satellite instability do not derive any bene t rom adjuvant

chemotherapy with 5- uorouracil (the mechanism o action

o uorouracil is described in Chapter 37) In contrast,

5- uorouracil is o en part o adjuvant chemotherapy or

microsatellite-stable colon tumors

Persons who are homozygous or compound heterozygous

or mutations in DNA MMR proteins not only develop

gas-trointestinal cancer but also have brain tumors and

hemato-logic malignancies in childhood T is disorder is called

constitutional MMR de ciency syndrome (CMMR-D).

T e nucleotide-excision repair (NER) pathway recognizes

distortions o the DNA double helix that arise rom

environ-mental insults (e.g., sunlight or smoking) or

chemotherapeu-tic agents (e.g., platinum drugs) In addition, it repairs

lesions that lead to the stalling o transcription A section o

~30 nucleotides o one DNA strand is removed in one piece

and then resynthesized

NER can be divided into two subpathways: global genome

(GG) repair and transcription-coupled ( C) repair GG-NER

occurs throughout the genome when helix-distorting lesions

(e.g., interstrand crosslinks) are recognized By contrast,

C-NER occurs when RNA polymerases, which negotiate

helix-destabilizing lesions ine ciently, become stalled on the

DNA In C-NER, the transcribed strand o the DNA is

repaired more e ciently than the nontranscribed strand A

network o histone-modi ying processes appears to assist

access to histone-bound DNA GG-NER and C-NER

acti-vate the same set o NER enzymes to repair the DNA

Distortions o DNA helices, recognized by GG-NER, are a

hallmark o many types o DNA damage GG-NER deals

mostly with intrastrand DNA adducts and crosslinks

Common intrastrand DNA adducts include the ollowing

lesions: (1) -, C-, C -, and CC-cyclobutane dimers that

are caused by ultraviolet (UV) light (either rom the sun or

tanning lights; Fig 2.6); (2) adducts between adenine or

guanine and polycyclic aromatic hydrocarbons ( ound in

cigarette smoke and environmental contaminants; Fig 2.7);

and (3) adducts between GG or AG sequences and platinum

drugs (e.g., cisplatin, carboplatin, or oxaliplatin, which are

used in chemotherapy or solid tumors; Figs 2.8 and 2.9)

Fig 2.6 UV lig ht induc e s intras trand c ro s s linking o f dine bas e s into c yc lobutane dime rs

pyrimi-H

N

Comple me nta ry DNA stra nds in one double he lix

H

O O

N

Trang 31

when exposed to UV light, and they also have an increased susceptibility to cancer that results rom smoking or carcino-gens in the diet Cockayne syndrome is characterized by ema-ciation and short stature as well as neurological impairment,

o en also by photosensitivity richothiodystrophy is terized by brittle hair and sometimes also by photosensitivity

charac-T ese disorders dramatically reveal the importance o nents o the NER system

compo-Inadequate repair o drug-induced damage is taken

advan-tage o in the treatment o cancer esticular cancer cells, or

instance, have a low capacity or NER and thus readily undergo programmed cell death (see Section 5 and Chapter 8) when exposed to platinum drugs T is drug sensitivity is a major reason or the high cure rate o testicular cancer that is

achieved with therapy that includes cisplatin (see Figs 2.8and 2.9)

T e NER pathway works together with HR (see Section 4.2)

to repair interstrand crosslinks, such as those generated by platinum compounds, nitrogen mustards, or psoralen

INTERSTRAND CROSSLINKS

Nondividing cells repair double-strand breaks chie y via NHEJ Dividing cells repair double-strand breaks and inter-strand crosslinks via a combination o NHEJ and HR repair

HR repair involves the copying o in ormation rom a nearby sister chromatid or homologous chromosome Patients who have hereditary def ciencies o HR repair have a variety o cancer syndromes

Ionizing radiation (e.g., in the orm o high-energy x-rays)

can give rise to single- and double-strand breaks Ionizing

radiation damages DNA directly or indirectly by orming DNA-damaging ree radicals (mostly hydroxyl radicals,

OH•, rom water) When ionizing radiation cuts both DNA strands within 10 to 20 base pairs o each other, the cuts gener-ate a double-strand break T e ends o the breaks o en contain an inappropriate phosphate group or a ragment o deoxyribose

In nondividing cells, double-strand breaks are largely

repaired by NHEJ (see Fig 2.11) T e proteins Ku70 (also called XRCC6) and Ku80 (also called XRCC5) bind to the

broken ends o the DNA T e Ku proteins recruit the

DNA-dependent protein kinase catalytic subunit and the nuclease

Artemis, which processes the ends o the strands i needed End processing may be accompanied by a loss o nucleotides

As needed, the DNA polymerases λ and µ then insert tides with or without a template A DNA ligase complex (con-

nucleo-sisting o XLF, XRCC4, and DNA ligase IV) then ligates the ends o the DNA; this ligase complex tolerates some gaps and mismatches (In contrast to double-strand breaks, single-strand breaks are repaired by BER; see Section 1.)

NHEJ can introduce mutations and is there ore a tially tumorigenic process Nonetheless, these mutations are

poten-carcinomas, squamous cell poten-carcinomas, and melanomas o

the skin (see Fig 2.6) Inadequate repair o smoking-induced

damage plays a role in the development o lung cancer

(see Fig 2.7)

Debilitating heritable de ciencies in NER are seen in the

rare autosomal recessively inherited diseases xeroderma

pig-mentosum, Cockayne syndrome, and a orm o light-sensitive

trichothiodystrophy (all occur in less than 1 in 100,000

people) All these diseases can, in turn, be subdivided into

several types, depending on the protein that is mutated

Patients with xeroderma pigmentosum readily develop tumors

Fig 2.7 Adduct of the principal metabolite of the carcinogen dibenzo[a,l]

pyrene with deoxyadenos ine in DNA

De oxya de nos ine

Bro nc ho g e nic c arc ino ma,

s quamo us c e ll type

Dibe nzo [a ,l]pyre ne :

H2C

N N

N

NH

OH HO

HO

N O O O

Trang 32

pathogenic protein production As described in Chapter 8, an increased rate o mutations paves the way or the development

o a tumor

Intense irradiation o cells with ionizing radiation

pro-duces such extensive and persistent DNA damage that af ected, heavily damaged cells undergo programmed cell death; this is

the basis o radiation therapy o tumors Radiation therapy

thought to be less damaging to cells than unrepaired DNA

double-strand breaks because unprotected ends at the

break-point would be degraded Furthermore, some o the

unre-paired DNA segments would lack centromeres or telomeres,

which would be catastrophic or the genome o a cell NHEJ

can take various paths even with the same starting damage

Mutations caused by NHEJ o en consist o one to 10

nucleo-tide deletions or three or ewer nucleonucleo-tide insertions When

a nucleus contains numerous double-stranded DNA

rag-ments, NHEJ even carries a risk o joining the wrong DNA

ragments, which results in translocation (material rom one

chromosome is joined to another chromosome) T is can

cause a protein-coding segment o a gene to be controlled by

a promoter that induces aberrant transcription and thus

Fig 2.8 Cis platin-induc e d intras trand c ro s s linking be twe e n two adjac e nt g uanine bas e s

A, Cis platin B, Cis platin adduct with guanine bas es in DNA C, Solution s tructure of a cis platin-DNA

intra-s trand crointra-s intra-s link Platination cauintra-s eintra-s partial unwinding of the double helix, an unuintra-s ual angle of the planeintra-s of

the guanine bas es , and an overall bend in the long axis of the helix Platinum drugs als o generate inters trand

cros s links , which have to be repaired via homologous recombination repair (s ee Section 4.2 ) (Bas ed on Protein Data Bank [ www.rcs b.org ] 1A84 from Gelas co A, Lippard SJ NMR s olution s tructure of a DNA dodecamer duplex containing a cis -diammineplatinum [II] d[GpG] intras trand cros s -link, the major adduct

of the anticancer drug cis platin Biochem istry 1998;37:9230–9239.)

O O P

N

NH HN

NH2

H2N

NH3

NH3Cl

Pt Cl

O O

Fig 2.9 Us e o f c is platin in the tre atme nt o f te s tic ular c anc e r.

Patients us ually undergo orchidectomy and then often receive adjuvant

chemotherapy with cis platin Treatment is s ucces s ful in part largely

becaus e the tumor cells have a low capacity for nucleotide-excis ion

repair and then undergo apoptos is

Norma l te stis S e minoma (cut)

Fig 2.10 Nuc le o tide e xc is io n re pair o f intras trand c ro s s links The cros s links can be TT-, CT-, or CC-cyclobutane dimers (UV induced; see Fig 2.6 ), s ingle nucleotides linked to polycyclic aromatic compounds (as found in cigarette s moke; s ee Fig 2.7 ), or platinum- linked purine nucleotides (e.g., cis platin induced; s ee Fig 2.8 )

-Intras trand c ro s s link (dis to rts double he lix)

Damage re c o gnitio n pro te ins ,

he lic as e s , e ndo nuc le as e s

DNA po lyme ras e , DNA lig as e

Deoxyribonucle otide s

Trang 33

(Homo lo gy-Dire c te d Re pair)

HR repair, like NHEJ described above, repairs DNA

double-strand breaks HR most o en uses a sister chromatid as a

template or repair In contrast to NHEJ, HR is generally rate In the S and G2 phases o the cell cycle, double-strand breaks can arise mostly rom problems with DNA replication (see Chapter 3), such as unrepaired single-strand breaks or complexes o DNA with poisoned topoisomerase It is esti-

accu-mated that a normal dividing cell under physiological cumstances needs to repair ~50 double-strand breaks per cell cycle, and most o these breaks are handled by HR

cir-Interstrand crosslinks are repaired by NER alone (GG-Ner

and/or C-NER; see Section 3) or by an obligatory tion o HR repair and NER (Intrastrand crosslinks are repaired

combina-by NER.) Interstrand crosslinks result rom platinum drugs,

nitrogen mustards, or psoralens Platinum drugs (e.g.,

cispla-tin, carboplatin) and nitrogen mustards (e.g., phamide) are used in chemotherapy to kill tumor cells Psoralens (e.g., methoxypsoralen) are used or the treatment

cyclophos-o pscyclophos-oriasis and vitiligcyclophos-o UV light induces pscyclophos-oralens tcyclophos-o cyclophos-orm

cyclobutanes with staggered pyrimidine bases on the two strands o a DNA double helix Psoriasis (Fig 2.12) is a common skin disorder that is marked by the hyperproli era-tion o keratinocytes; treatment with a psoralen plus light reduces this hyperproli eration Vitiligo (see Fig 35.18) is a condition involving the patchy loss o skin pigmentation that

af ects 1% to 2% o the population T e loss o pigmentation

is due to an absence o melanin pigment-producing cells, which in turn may be due to in ammation (see Chapter 35) reatment with a psoralen plus light is ef ective and might work by diminishing in ammation Although the psoralens kill some cells as intended, they also increase the rate o muta-tion in other cells, which explains the side ef ect o an increased rate o skin cancer

aims not only to introduce double-strand breaks but also

addi-tional DNA lesions within the region o the break Cells that

have DNA with such clustered lesions are especially likely to

die

In the adaptive immune system, NHEJ is involved in

recombining V, D, and J segments o antibodies and -cell

receptors T e inaccuracies o NHEJ help increase the

diver-sity o antibodies and -cell receptors Patients who have a

de ciency in NHEJ can also have a de ciency in their adaptive

immune system

Fig 2.11 Re pair of radiation-induc e d double -s trand bre aks

by no nho mo lo g o us e nd jo ining Some radiation is natural

Radia-tion is als o a mains tay of cancer treatment

Intac t

do uble he lix

Do uble -s trand bre ak

Co rre c t re pair Re pair with e rrors

(e g , de le tio n)

X-rays

Re pair by no nho mo log ous e nd jo ining

Fig 2.12 Ps o rias is is s ome time s tre ate d with photore ac tive ps o rale ns , whic h c aus e DNA intras trand and inte rs trand c ro s s links

Trang 34

dominant ashion (as in Lynch syndrome; see Section 2) Heterozygosity or a de ective BRCA1 or BRCA2 allele is a

requent cause o the hereditary breast and ovarian cancer

syndrome (HBOCS).

Germline heterozygosity or a mutation in the PALB2 gene

is associated with an increased risk o pancreatic cancer and

breast cancer ogether, mutations in PALB2 and BRCA2 are

responsible or much o hereditary pancreatic cancer (other contributors are mutations in a gene or a MMR protein and mutations in the CDKN2A gene) PALB2 mutations are responsible or only a ew percent o patients who have heredi-tary breast cancer

Fanconi anemia is a heritable, rare syndrome that is

char-acterized by bone marrow ailure, resulting in anemia, penia, and thrombopenia, as well as mal ormations Af ected persons are at high risk o developing hematological malig-nancies and solid tumors Patients who have Fanconi anemia are homozygous, compound heterozygous, or hemizygous or

leuko-a mutleuko-ant protein o the Fleuko-anconi leuko-anemileuko-a network (leuko-also cleuko-alled

Fanconi anemia pathway) T e complete network consists o

at least 16 proteins that play a role in DNA repair PALB2 and BRCA2 are members o the Fanconi anemia network Patients

Fig 2.13 Appro ximate ly 10% o f wo me n have bre as t c anc e r during the ir life time , and abo ut 5% o f the s e patie nts inhe r- ite d o ne mutant BRCA alle le that e nc o de s a pro te in with impaire d func tio n. With time, the other, normal allele becomes non- functional, thereby impairing homologous recombination repair of DNA Cells without functioning homologous recombination repair accumulate mutations at an increas ed rate and are more likely to give ris e to a tumor

Patients can be tes ted for BRCA gene mutations

A er a double-strand break occurs, the MRN complex

(consisting o Mre11, Rad50, and Nbs1) binds to the ends o

the DNA and activates the signaling kinase A M (ataxia

tel-angiectasia mutated) A M, in turn, phosphorylates

numer-ous proteins, some o which halt the cell cycle, while others

increase the DNA repair activity T e MRN complex cuts one

DNA strand ~100 to 200 base pairs rom the break and then

resects this strand toward the break Another protein complex

resects the same strand in the opposite direction so that a

single-strand overhang is generated that can be greater than

1000 bp long One such overhang is generated in each piece

o broken DNA Meanwhile, MRN also tethers together the

ends o the two pieces o DNA T e BRCA1 protein (breast

cancer 1) orms a dimer with the PALB2 protein and recruits

the BRCA2 protein, which in turn helps load the recombinase

RAD51 RAD51 then osters the invasion o a sister chromatid

or a homologous chromosome by a 3′ overhang T e invaded

DNA strand serves as a template or an elongation o the 3′

overhang Subsequently, helicases and nucleases resolve the

entangled DNA strands

T e use o a homolog or HR repair leads to gene

conver-sion, which can be the cause o a loss o heterozygosity

(LOH) T e homologous chromosomes, derived rom the

mother and ather, contain similar but not identical sequences

Gene conversion re ers to the nding that the sequence o one

parental allele converts to the sequence o the other parental

allele Hence, a somatic cell with one unctional and one

unctional copy o a gene may give rise to a cell with two

non unctional copies LOH can also be used to describe the

deletion o the examined sequence T e term uniparental

disomy is applied when there is a loss o a chromosome or

chromosome segment (usually containing multiple genes)

rom one parent and a gain o the lost sequence rom the other

parental chromosome In patients who are heterozygous or a

de ciency o a tumor suppressor, HR repair may lead to the

complete loss o tumor suppressor activity, which may be

tumorigenic (see Chapter 8)

Impairment o the activity o the MRN complex or the

A M kinase leads to an increased rate o mutation, and to an

increased sensitivity toward therapeutic ionizing radiation

Ataxia-telangiectasia, seen in about one per 300,000 births,

is due to homozygosity or compound heterozygosity or

inac-tivating mutations in the A M gene Nijmegen breakage

syn-drome and ataxia-telangiectasia–like disease are much rarer

diseases that are due to homozygosity or compound

hetero-zygosity or inactivating mutations that af ect the MRN

complex Reduced amounts o the MRN complex are also

observed in about 20% o breast tumors.

About 5% o women who have breast cancer (Fig 2.13), or

~0.1% o all men and women, have inherited a mutation in

the BRCA1 or BRCA2 genes Over time, the remaining,

normal BRCA allele becomes lost or inactivated Without

unctional BRCA proteins, cells accumulate DNA alterations

at an increased rate and are thus prone to tumorigenesis

Patients with a heritable BRCA mutation are at an increased

risk or breast cancer, ovarian cancer, and other tumors T e

propensity or tumor ormation is inherited in autosomal

Trang 35

various means, the checkpoint kinases lead to a halt in the cell cycle by blocking the G1/S transition, S phase, the G2/M tran-sition, or M phase (see Section 1 in Chapter 8) T is allows DNA repair pathways, including translesion DNA synthesis,

to repair DNA damage

DNA repair pathways are o en redundant: although a ticular type o damage is typically repaired mostly by one pathway, it can o en be repaired by an alternative pathway I DNA damage is repaired, the signal blocking the cell cycle is eliminated, and progression through the cycle resumes I the damage is not repaired, the DNA damage signal persists and can trigger apoptosis

par-Some chemotherapeutic drugs kill cells by inducing DNA

damage that is so overwhelming that the cells undergo tosis T e sensitivity o normal and abnormal cells to chemotherapy-induced DNA alterations depends on many variables, including drug uptake and e ux, the capacity or DNA repair, the ability o cells to sense and transduce the DNA damage response, and the likelihood that DNA damage leads to apoptosis Many tumor cells have altered sensitivity

apop-to DNA damage-induced apopapop-tosis Cells that survive damage rom chemotherapeutic drugs (e.g., because entry into apop-tosis is misregulated or DNA damage-sensing mechanisms ail

to detect the damage) may give rise to a new tumor or vate the behavior o the existing tumor

aggra-Intense ionizing radiation, such as that used or radiation

therapy o cancer, causes cell death not just by the sheer volume o damage to DNA, but also by clustering damage within one to two turns o the DNA helix It is unclear why clustered damage is particularly lethal

who have two mutant BRCA2 alleles have the D1-type o

Fanconi anemia, and patients who have two mutant PALB2

alleles have the N-type Protein complexes o the Fanconi

anemia network coordinate some o the DNA excision, strand

invasion, and resolution o HR In the general population,

several types o sporadic tumors are de cient in one o the

proteins o the Fanconi anemia network, which bears on the

susceptibility o these tumors to DNA crosslinking agents

Patients who have Fanconi anemia are hypersensitive to

ion-izing radiation and DNA crosslinking agents (e.g., cisplatin or

cyclophosphamide)

HR not only plays a role in DNA repair but also in orming

crossovers in meiosis or the purpose o identi ying and

pairing homologous chromosomes, thereby increasing genetic

diversity among of spring

CYCLE AND REGULATES APOPTOSIS

As is outlined in Chapter 8, cancer is the result o damage to

the genome such that cell growth and survival are no longer

properly regulated Inadequate DNA repair increases the rate

o mutation and thus avors the ormation o a tumor Cells

have means o assessing DNA damage and determining

whether to survive or sel -destruct

A cell’s DNA damage response senses DNA damage, slows

progression through the cell cycle, and coordinates this with

DNA repair; when DNA damage is persistent, the response

can also initiate apoptosis Most DNA repair pathways are

discussed in Sections 1 to 4 ranslesion DNA synthesis is

discussed in Section 2 o Chapter 3 Fig 2.14 provides an

overview o these repair pathways

T e DNA damage response is best studied in cells that

contain double-strand breaks Such breaks eventually lead to

the activation o the kinases A R and A M, which in turn

activate the checkpoint kinases Chk1 and Chk2 T rough

Fig 2.14 Ove rvie w o f DNA re pair pathways DNA replication and trans les ion DNA s ynthes is are explained in Chapter 3

Ba s e -e xcis ion re pa ir, mis ma tch re pa ir, nucle otide -

e xcis ion re pa ir

DNA s ingle -s tra nd da ma ge

DNA re plica tion, s ta lls

DNA double-s trand bre a ks

Unre pa ire d single -strande d

DNA dama ge

Deamination, hydroxylation, methylation, or loss of a base; damage to a deoxyribose; base mismatch;

intrastrand crosslinks;

repa ir

Platinum drugs, nitrogen

mustards, psoralens + light

Trang 36

FURTHER READING

■ Brenerman BM, Illuzzi JL, Wilson III DM Base excision repair capacity in in orming healthspan Carcinogenesis 2014;35:2643-2652

■ Erie DA, Weninger KR Single molecule studies o DNA mismatch repair DNA Repair (Amst) 2014;20:71-81

■ La rance-Vanasse J, Williams GJ, ainer JA Envisioning the dynamics and exibility o Mre11-Rad50-Nbs1 complex

to decipher its roles in DNA replication and repair Prog Biophys Mol Biol 2015;117:182-193

■ Lindahl Instability and decay o the primary structure o DNA Nature 1993;362:709-715

■ Marteijn JA, Lans H, Vermeulen W, Hoeijmakers JH Understanding nucleotide excision repair and its roles in cancer and ageing Nat Rev Mol Cell Biol 2014;15:465-481

■ Mehta A, Haber JE Sources o DNA double-strand breaks and models o recombinational DNA repair Cold Spring Harb Perspect Biol 2014;6(9):a016428

properties o DNA, the destructive ef ects o other cellular

constituents, or ionizing radiation T e short patch repair

pathway replaces a single damaged nucleotide, while the

long-patch repair pathway replaces a stretch o 2 to 10

consecutive nucleotides T e complementary DNA strand

serves as a template or the insertion o nucleotides

■ MU YH-associated polyposis (MAP) is caused by a

homo-zygous or compound heterohomo-zygous de ciency o DNA

MYH glycosylase, which plays a role in repairing

8-oxo-guanine (produced rom 8-oxo-guanine by a radical) T e disease

is associated with the ormation o numerous polyps in the

colon, as well as early colorectal cancer

■ DNA mismatch repair (MMR) processes tackle

single-base DNA mismatches and DNA loops, which arise rom

errors in the synthesis o DNA, rom the spontaneous

deamination o C to U or methyl-C to , or rom

homolo-gous recombination (HR) repair Enzymes o the DNA

MMR pathway degrade and resynthesize a portion o a

DNA strand, which is o en the most recently synthesized

strand

■ Lynch syndrome is due to an inherited MMR de ciency

Both inherited and acquired de ciencies in MMR can

cause cancer, especially o the colon, uterus, and ovaries

An MMR de ciency in tumor cells can o en be detected

as de cient immunohistochemical staining o MMR

pro-teins and by DNA microsatellite instability (MSI) (i.e., a

shortening o the lengths o certain nucleotide repeats)

■ Nucleotide-excision repair (NER) involves the removal and

appropriate replacement o a contiguous stretch o ~24 to

32 nucleotides around a helix-distorting lesion on one

strand o a DNA double helix Such lesions commonly stem

rom the crosslinking o the pyrimidine bases ( or C) by

exposure to UV light, rom the reaction o metabolites o

polycyclic aromatic hydrocarbons ( ound in smoke) with

purine bases (A or G), or rom the crosslinking o adjacent

purine bases by platinum drugs (used in chemotherapy o

tumors) A high rate o production o helix-distorting DNA

lesions is associated with an increased risk o cancer (e.g.,

melanoma and lung cancer)

■ A er a double-strand break, HR involves resection o one

strand to produce a long single-strand overhang, invasion

o the homologous chromatid, copying o the in ormation,

and separation o the two chromatids

■ Ionizing radiation (e.g., high-energy x-rays) causes

double-strand breaks that can be repaired by NHEJ or by HR

■ T e therapeutically used platinum drugs, nitrogen

mus-tards, and photoreactive psoralens produce interstrand

crosslinks that can be repaired by HR repair T e BRCA1

and BRCA2 proteins participate in HR repair Mutations in

the BRCA genes can convey increased susceptibility to

breast and ovarian cancer

■ DNA damage-sensing pathways can halt the cell cycle to

allow time or DNA repair Cells with excessive unrepaired

DNA damage o en undergo apoptosis Some o the drugs

used in the chemotherapy o cancer kill cells by causing

DNA damage in excess o the cells’ capacity or repair

T ese drugs are inherently mutagenic to all cells

Re vie w Que s tio ns

1 A 48-year-old woman has endometrial cancer and goes a hysterectomy Immunohistochemistry o tumor tissue reveals the presence o MLH1 and PMS2, but an absence o MSH2 and MSH6 Based on this nding, the most likely diagnosis is which o the ollowing?

T e adduct leads to distortion o the DNA double helix

T e lesion is most likely repaired by which one o the lowing DNA repair pathways?

ol-A Homologous recombination repair

Trang 37

4 A atoxin is a polycyclic aromatic hydrocarbon that is

pro-duced by Aspergillus species, which o en grow on cereals,

peanuts, and nuts T e liver converts ingested a atoxin to

a compound that reacts with guanine in DNA A stable

adduct o guanine and the a atoxin derivative is

predomi-nantly repaired by which one o the ollowing DNA repair

A (1) Deamination o C to U and (2) mismatch repair

B (1) Double-strand break and (2) homologous

Trang 38

SYNOPSIS

■ During DNA replication, each DNA strand serves as a template

for the synthesis of a new, complementary DNA strand

Replica-tion starts at many sites on each chromosome As

double-stranded DNA is opened up for replication, each strand can be

copied continuously in one direction, but it must be copied in

many small segments in the opposite direction.

■ Translesion DNA polymerases help DNA replication continue

through unrepaired DNA lesions.

■ Telomeres, the ends of chromosomes, shorten with each round

of replication This shortening plays a role in senescence Cells

of the germline and stem cells use telomerase to keep the

length of their telomeres constant.

Replication o a DNA double helix is semiconservative:

each strand o an existing DNA double helix serves as a plate or the synthesis o a new complementary strand At the end o this process, each o the two double helices contains one o the old DNA strands and one o the newly synthesized DNA strands (Fig 3.1)

tem-During replication, DNA synthesis proceeds in a 5′ to 3′ direction (Fig 3.2) T e 3′ hydroxyl group at the end o a growing DNA strand per orms a nucleophilic attack on the phosphorus atom o the incoming deoxyribonucleoside tri-phosphate that is closest to the sugar (i.e., the α-phosphate o the incoming dN P) I the nucleotide at the 3′ end o a DNA strand lacks the 3′ hydroxyl group, the DNA strand cannot be elongated

Replication can start at thousands o predetermined

regions, the origins o replication (ORIs) ORIs are spaced

~50,000 to 300,000 base pairs apart T e use o these origins

is regulated T ere are early- and late-replicating origins thermore, most origins are never used under normal circum-stances (i.e., they are dormant) Dormant origins can become active when replication stalls

Fur-T e ollowing processes ensure that the DNA around each ORI is replicated only once per cell cycle: During the G1 phase

o the cell cycle, just be ore S phase (see Chapter 8),

multipro-tein origin-recognition complexes assemble on the ORIs in

a process termed licensing T is happens only in the presence

o loading actors, which in turn are present only during this G1 phase In the G1-to-S-phase transition and during S phase (when loading actors are no longer present), an A P-driven helicase in the assembled complex is activated, and the complex departs rom the ORI T e activity o cyclin-dependent kinases (CDKs; see Chapter 8) is high as cells exit rom G1 and enter S phase, and it remains high until chromo-some segregation takes place during M phase T is high CDK activity inhibits licensing during the S and M phases, thus preventing rereplication

Starting at an ORI, separation o the complementary DNA

strands gives rise to two replication orks (see Fig 3.3) As

A P-driven helicases separate double-helical DNA into single

strands, single-strand–binding proteins such as RPA

(repli-cation protein A) partially wrap around the single-stranded

DNA and prevent the hybridization o bases within the same

strand or with the complementary DNA strand

opoisomer-ase I removes the superhelical stress rom the DNA (see

Chapter 1) Each strand o the DNA double helix is read in a 3′→5′ direction, giving rise to new, complementary DNA

strands that are called leading strands (see Figs 3.3 and 3.5)

3

■ Outline the replication of DNA.

■ Describe the factors that contribute to the delity of DNA

replication.

■ Describe the structure of telomeres, explain how replication

leads to shortening of telomeres, and describe how select cells

maintain an adequate length of their telomeres.

During replication, each strand o a DNA double helix serves

as a template or the synthesis o a new comple mentary DNA

strand opoisomerase I releases the superhelical strain and

helicases catalyze the separation o complementary DNA

strands Replication o a section o DNA starts with the

syn-thesis o an RNA primer T en, DNA polymerase catalyzes

the addition o deoxyribonucleotides Finally, the RNA

primer is replaced by deoxyribonucleotides

T e packing o nuclear DNA into nucleosomes, 30-nm

chromatin bers, and higher-order structures is described in

Chapter 1 In preparation or DNA replication (i.e., the

copying o the genome), higher-order DNA structures are

dismantled by chromatin remodeling actors Such actors

include enzymes that modi y proteins in chromatin (e.g.,

acetylases and deacetylases, methylases and demethylases,

kinases, and phosphatases) and proteins that replace existing

proteins in chromatin

Nuclear DNA is replicated during S phase o the cell cycle

(see Chapter 8), which usually takes a ew hours

Mitochon-drial DNA is replicated on demand, which can occur

indepen-dently o the replication o nuclear DNA T e ollowing is an

account o DNA replication in the nucleus

Trang 39

DNA Replication 23

As replication proceeds, an increasing length o DNA on the 3′ side o the ORI remains uncopied, because the template can

be read only in the 3′→5′ direction Once approximately 100

to 200 uncopied bases are exposed, a DNA polymerase works

on these strands as well, producing 100 to 200 nucleotide-long

pieces o DNA that are called Okazaki ragments (see Figs.3.3 and 3.5) T e Okazaki ragments are eventually ligated,

and this strand is called the lagging strand T us, synthesis o

the leading strand is continuous, while synthesis o the lagging strand is discontinuous

DNA polymerase α is a multisubunit enzyme complex that

contains a DNA polymerase and an RNA polymerase It uses ribonucleoside triphosphates (i.e., A P, C P, G P, U P) to

synthesize a complementary RNA primer that is ~7 to 12

nucleotides long (Figs 3.4, 3.5) T e DNA polymerase then extends the RNA primer by ~20 nucleotides All RNA and DNA is synthesized by the addition o a nucleoside 5′-triphos-phate to the 3′-hydroxyl group o the preceding nucleotide (i.e., the newly synthesized strand grows in a 5′→3′ direction) Neither the RNA polymerase or the DNA polymerase in the DNA polymerase α complex can carry out proo reading; the complex there ore incorporates noncomplementary nucleo-tides at a higher requency than DNA polymerase δ (see below)

DNA polymerase δ (the DNA polymerase responsible or

the bulk o nucleotide incorporation) and DNA polymerase

ε (which plays a minor role not shown in Fig 3.5) elongate the strand synthesized by DNA polymerase α Each poly-merase has a proo reading unction; i the bases o the growing DNA strand do not match the template, the enzyme stalls, excises the mismatched nucleotide, and then continues polymerization

When DNA polymerase δ reaches an RNA primer on the lagging strand, the primer and up to ~30 deoxyribonucleo-tides that ollow it are displaced and excised DNA polymerase

δ inserts the missing complementary nucleotides, and the

DNA ligase joins the 3′ end o the most recently synthesized

piece o DNA with the appropriate 5′-end o the previously synthesized piece o DNA

DNA replication has a very high delity On average, each

replication o the human genome (involving ~3 billion base pairs) introduces only about three base changes T e high accuracy is in large part due to the substrate speci city

Fig 3.1 DNA re plic atio n is s e mic o ns e rvative

+ Nucle otide s

Re plic atio n

Fig 3.2 DNA re plic atio n o c c urs 5 ′ to 3 ′ and is s to ppe d by

inc o rpo rate d s ynthe tic dide o xyribo nuc le otide s that lac k the

3′ hydro xyl g ro up. Dideoxyribonucleotides are us ed clinically to treat

cancer and in the laboratory for DNA s equencing and various DNA-bas ed

CH 2

H

H H

O OH

CH 2

H

H H

O H

Ba s e n

O O

5´

O–P

O O

O–P

O

O–P O

CH 2

H

H H

O OH

3´ e nd 5´ e nd

Origin o f Re plic ation

Fig 3.4 Prime r s ynthe s is during DNA re plic atio n.

RNA prime r

Trang 40

lamivudine also acts as a chain terminator Lamivudine is used

in the treatment o hepatitis B and human immunode ciency virus-1

Arabinosylcytosine and udarabine (Fig 3.7) inter ere with DNA replication, and both drugs are used or the

treatment o acute leukemias Inside cells, these drugs are

phosphorylated DNA polymerase incorporates tosine triphosphate and udarabine triphosphate into DNA However, these synthetic nucleotides are poor substrates or excision and replacement, as well as or continued replication

arabinosylcy-T e decrease in replication leads to double-strand breaks In addition, udarabine at the 3′ terminus o a piece o DNA prevents ligation by DNA ligase, leading to persistent single-strand breaks (i.e., nicked DNA) Once a cell has incorporated

Fig 3.5 DNA re plic atio n, s ho wing the le ading and lag g ing s trand at a re plic atio n fo rk.

DNA

DNA (c o pie d unre liably by DNA polyme ras e a ) He lic as e s e pa ra te s

comple me nta ry DNA s tra nds ;

topo is o me ras e re lie ve s tors iona l

s tre s s from s upe rhe lica l winding

RNA prime r (s ynthe s ized by DNA polyme ras e a )

Re plic atio n pro te in A

DNA (c o pie d ac c urate ly by DNA po lyme ras e d)

To be re plac e d by DNA po lyme ras e d

Okazaki frag me nt

(100–200 nucle otide s )

Fig 3.6 Drug s that pre fe re ntially inhibit DNA s ynthe s is in

re tro virus e s Zidovudine is an analog of thymidine, and lamivudine is

an analog of deoxycytidine Res idues that differ from the phys iological nucleos ide are s hown in red

NH O

S

and proo reading unction o DNA polymerase δ, as well as

the e ciency o postreplication DNA mismatch repair (see

Section 2 in Chapter 2)

Following replication, DNA is assembled into nucleosomes

and higher-order chromatin structures using chromatin

assembly actors, including existing and newly synthesized

histones

DNA polymerases and DNA ligases rom a variety o

organisms are used or in vitro DNA diagnostic methods (see

Chapter 4)

Dideoxyribonucleotides that inter ere with DNA

replica-tion are used in cancer chemotherapy, as antiviral drugs, in

DNA diagnostics, and in Sanger-type DNA sequencing (see

Chapter 4) Nucleotides without a 3′-hydroxyl group (e.g.,

ddA P, ddG P, ddC P, and dd P), can be incorporated

into the DNA, but because they lack a 3′-hydroxyl group, they

are chain terminators (see Fig 3.2)

Zidovudine and lamivudine (Fig 3.6) both inhibit viral

reverse transcriptases (enzymes that copy viral RNA into

DNA) but have only a minor ef ect on human DNA

polymer-ases Both drugs are used against in ections with retroviruses

(i.e., viruses that contain an RNA genome) Zidovudine is an

analog o thymidine Enzymes in the cell convert zidovudine

to the triphosphate orm, and reverse transcriptase

subse-quently incorporates it into growing DNA strands T ese

DNA strands cannot be elongated because zidovudine lacks a

3′-hydroxyl group Lamivudine likewise is phosphorylated

inside cells and then markedly inhibits viral reverse

transcrip-tases but not human DNA polymerases Like zidovudine,

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