biochemistry and Genetic science 2018.pdf

HY LY MY HY LY MY HIGH YIELD LOW YIELD MEDIUM YIELD High-Yield LOW YIELD MEDIUM YIELD REINFORCEMENT FUNDAMENTALS REINFORCEMENT FUNDAMENTALS 1... Comparison of Gene Expression and DNA Rep

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Assistant Professor, Department of Family Medicine and Community Health

University of Minnesota Medical School, Duluth Campus

Duluth, MN

Part I: Biochemistry

Chapter 1: Nucleic Acid Structure and Organization                  3

Chapter 2: DNA Replication and Repair                           17

Chapter 3: Transcription and RNA Processing                       33

Chapter 4: The Genetic Code, Mutations, and Translation             49

Chapter 5: Regulation of Eukaryotic Gene Expression                75

Chapter 6: Genetic Strategies in Therapeutics                      87

Chapter 7: Techniques of Genetic Analysis                        103

Chapter 8: Amino Acids, Proteins, and Enzymes                   119

Chapter 9: Hormones                                         135

Chapter 10: Vitamins                                          149

Chapter 11: Energy Metabolism                                 163

Chapter 12: Glycolysis and Pyruvate Dehydrogenase               175

Chapter 13: Citric Acid Cycle and Oxidative Phosphorylation         193

Chapter 14: Glycogen, Gluconeogenesis, and the Hexose

Monophosphate Shunt                              205Chapter 15: Lipid Synthesis and Storage                          223

Chapter 16: Lipid Mobilization and Catabolism                    243

Chapter 17: Amino Acid Metabolism                             265

Chapter 18: Purine and Pyrimidine Metabolism                    289

Table of Contents

Chapter 2: Population Genetics                                  333Chapter 3: Cytogenetics                                        347Chapter 4: Genetics of Common Diseases                         371Chapter 5: Recombination Frequency                             379Chapter 6: Genetic Diagnosis                                    391Index                                                              407

Additional resources available at www.kaptest.com/usmlebookresources

PART I

Nucleic Acid Structure

and Organization

Learning Objectives

❏ Explain information related to nucleotide structure and nomenclature

❏ Use knowledge of organization of DNA versus RNA

❏ Understand general features of a chromosome

CENTRAL DOGMA OF MOLECULAR BIOLOGY

An organism must be able to store and preserve its genetic information, pass

that information along to future generations, and express that information as it

carries out all the processes of life The major steps involved in handling genetic

information are illustrated by the central dogma of molecular biology. 

Figure I-1-1.Central Dogma of Molecular Biology

DNATranscriptionReplication

Reversetranscription

Translation

Figure I-1-1 Central Dogma of Molecular Biology

Genetic information is stored in the base sequence of DNA molecules

Ultimately, during the process of gene expression, this information is used to

synthesize all the proteins made by an organism. 

Classically, a gene is a unit of the DNA that encodes a particular protein or RNA

molecule Although this definition is now complicated by our increased appreciation

of the ways in which genes may be expressed, it is still useful as a working definition

Gene Expression and DNA Replication

Gene expression and DNA replication are compared below Transcription, the

first stage in gene expression, involves transfer of information found in a

dou-ble-stranded DNA molecule to the base sequence of a single-stranded RNA

molecule If the RNA molecule is a messenger RNA, then the process known as

translation converts the information in the RNA base sequence to the amino

acid sequence of a protein

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When cells divide, each daughter cell must receive an accurate copy of the genetic information DNA replication is the process in which each chromosome

is duplicated before cell division

Table I-1-1 Comparison of Gene Expression and DNA Replication

Produces all the proteins an organism requires

Duplicates the chromosomes before cell division

Transcription of DNA: RNA copy of a small section of a chromosome (average size of human gene,

Occurs during S-phase

Translation of RNA (protein synthesis) occurs in the cytoplasm throughout the cell cycle

Replication in nucleus

The concept of the cell cycle can be used to describe the timing of some of these events in a eukaryotic cell The M phase (mitosis) is the time in which the cell divides to form 2 daughter cells Interphase describes the time between 2 cell divisions or mitoses Gene expression occurs throughout all stages of inter-phase Interphase is subdivided as follows:

• G1 phase (gap 1) is a period of cellular growth preceding DNA sis Cells that have stopped cycling, such as muscle and nerve cells, are said to be in a special state called G0

synthe-• S phase (DNA synthesis) is the period of time during which DNA replication occurs At the end of S phase, each chromosome has doubled its DNA content and is composed of 2 identical sister chroma-tids linked at the centromere

• G2 phase (gap 2) is a period of cellular growth after DNA synthesis but preceding mitosis Replicated DNA is checked for any errors before cell division

Chapter 1 ● Nucleic Acid Structure and Organization

Control of the cell cycle is accomplished at checkpoints between the various

phases by strategic proteins such as cyclins and cyclin-dependent kinases These

checkpoints ensure that cells will not enter the next phase of the cycle until the

molecular events in the previous cell cycle phase are concluded

Reverse transcription, which produces DNA copies of an RNA, is more

com-monly associated with life cycles of retroviruses, which replicate and express

their genome through a DNA intermediate (an integrated provirus) Reverse

transcription also occurs to a limited extent in human cells, where it plays a role

in amplifying certain highly repetitive sequences in the DNA (Chapter 7)

NUCLEOTIDE STRUCTURE AND NOMENCLATURE

Nucleic acids (DNA and RNA) are assembled from nucleotides, which consist

of 3 components: a nitrogenous base, a 5-carbon sugar (pentose), and phosphate

Five-Carbon Sugars

Nucleic acids (as well as nucleosides and nucleotides) are classified according to

the pentose they contain If the pentose is ribose, the nucleic acid is RNA

(ribo-nucleic acid); if the pentose is deoxyribose, the (ribo-nucleic acid is DNA

(deoxyribo-nucleic acid)

Bases

There are 2 types of nitrogen-containing bases commonly found in nucleotides:

purines and pyrimidines

Thymine

CH3

HNH

OH

Figure I-1-3 Bases Commonly Found in Nucleic Acids

• Purines contain 2 rings in their structure The purines commonly

found in nucleic acids are adenine (A) and guanine (G); both are found

in DNA and RNA Other purine metabolites, not usually found in

nucleic acids, include xanthine, hypoxanthine, and uric acid

• Pyrimidines have only 1 ring Cytosine (C) is present in both DNA and

RNA Thymine (T) is usually found only in DNA, whereas uracil (U) is

found only in RNA

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Nucleosides and Nucleotides

Nucleosides are formed by covalently linking a base to the number 1 carbon of

a sugar The numbers identifying the carbons of the sugar are labeled with

“primes” in nucleosides and nucleotides to distinguish them from the carbons

of the purine or pyrimidine base

Figure I-1-4.Examples of Nucleosides

HNN

NN

Figure I-1-4 Examples of Nucleosides

Nucleotides are formed when 1 or more phosphate groups is attached to the 5′

carbon of a nucleoside Nucleoside di- and triphosphates are high-energy pounds because of the hydrolytic energy associated with the acid anhydride bonds

com-Figure I-1-5 Examples of Nucleotides

O

NN

OO:

Figure I-1-5 Examples of Nucleotides

Figure I-1-6 High-Energy Bonds in a

Nucleoside Triphosphate

Figure I-1-6 High-Energy Bonds in

a Nucleoside Triphosphate

ON

NNN

NH2

CH2

OH OH

OPOO–

OPOO–

OPOO–

O

High-energybonds

ATP

Chapter 1 ● Nucleic Acid Structure and Organization

The nomenclature for the commonly found bases, nucleosides, and nucleotides

is shown below Note that the “deoxy” part of the names deoxythymidine, dTMP,

etc., is sometimes understood and not expressly stated because thymine is

almost always found attached to deoxyribose

Table I-1-2 Nomenclature of Important Bases, Nucleosides, and Nucleotides

Base Nucleoside Nucleotides

Nucleic acids are polymers of nucleotides joined by 3′, 5′-phosphodiester bonds;

that is, a phosphate group links the 3′ carbon of a sugar to the 5′ carbon of the

next sugar in the chain Each strand has a distinct 5′ end and 3′ end, and thus

has polarity A phosphate group is often found at the 5′ end, and a hydroxyl

group is often found at the 3′ end

The base sequence of a nucleic acid strand is written by convention, in the 5′→3′

direction (left to right) According to this convention, the sequence of the strand

on the left in Figure I-1-7 must be written 5′-TCAG-3′ or TCAG:

• If written backward, the ends must be labeled: 3′-GACT-5′

• The positions of phosphates may be shown: pTpCpApG

• In DNA, a “d” (deoxy) may be included: dTdCdAdG

In eukaryotes, DNA is generally double-stranded (dsDNA) and RNA is

gener-ally single-stranded (ssRNA) Exceptions occur in certain viruses, some of

which have ssDNA genomes and some of which have dsRNA genomes

Behavioral Science/Social Sciences

Figure I-1-7 Hydrogen-Bonded Base Pairs in DNA

N N

H3C

O

O O

O 5´CH 2

5´CH2

CH 3

3´

O 3´

O 3´

5´CH23´

O P O O O

O

P O

T

N

N N

N

A

N H

O

O O

O

5´CH2

P O O

O

O 3´

O 5´CH2

P O O O

O

O

O

O P O O H

N

O O

N

5´CH23´

O 3´

O 3´

C

N

N N

O

G

N H N H

N N

N N

H

O

O P O

5´CH 2 O

O P O

5´CH2O

N H H

H H

Chapter 1 ● Nucleic Acid Structure and Organization

DNA Structure

Some of the features of double-stranded DNA include:

• The 2 strands are antiparallel (opposite in direction)

• The 2 strands are complementary A always pairs with T (2 hydrogen

bonds), and G always pairs with C (3 hydrogen bonds) Thus, the base

sequence on one strand defines the base sequence on the other strand

• Because of the specific base pairing, the amount of A equals the

amount of T, and the amount of G equals the amount of C Thus, total

purines equals total pyrimidines These properties are known as

Chargaff’s rules

With minor modification (substitution of U for T) these rules also apply to

dsRNA

Most DNA occurs in nature as a right-handed double-helical molecule known

as Watson-Crick DNA or B-DNA The hydrophilic sugar-phosphate backbone

of each strand is on the outside of the double helix The hydrogen-bonded base

pairs are stacked in the center of the molecule There are about 10 base pairs per

complete turn of the helix A rare left-handed double-helical form of DNA that

occurs in G-C–rich sequences is known as Z-DNA The biologic function of

Z-DNA is unknown, but may be related to gene regulation

AT AT CG GC

TA GC CG AT

AT TA GC TA

GC GC AT TA

AT AT

Major Groove

Provide binding sites for regulatory proteins

Minor Groove

Figure I-1-8 The B-DNA Double Helix Figure I-1-8 B-DNA Double Helix

Note

Using Chargaff’s Rules

In dsDNA (or dsRNA) (ds = double-stranded)

Bridge to Pharmacology

Daunorubicin and doxorubicin are antitumor drugs that are used in the treatment of leukemias They exert their effects by intercalating between the bases of DNA, thereby interfering with the activity of topoisomerase II and preventing proper replication of the DNA

Other drugs, such as cisplatin, which

is used in the treatment of bladder and lung tumors, bind tightly to the DNA, causing structural distortion and malfunction

Behavioral Science/Social Sciences

Denaturation and Renaturation of DNA

Double-helical DNA can be denatured by conditions that disrupt hydrogen bonding and base stacking, resulting in the “melting” of the double helix into two single strands that separate from each other No covalent bonds are broken

in this process Heat, alkaline pH, and chemicals such as formamide and urea are commonly used to denature DNA

Denatured single-stranded DNA can be renatured (annealed) if the denaturing condition is slowly removed For example, if a solution containing heat- denatured DNA is slowly cooled, the two complementary strands can become base-paired again (Figure I-1-9)

Such renaturation or annealing of complementary DNA strands is an important step in probing a Southern blot and in performing the polymerase chain reaction (reviewed in Chapter 7) In these techniques, a well-characterized probe DNA

is added to a mixture of target DNA molecules The mixed sample is denatured and then renatured When probe DNA binds to target DNA sequences of suffi-cient complementarity, the process is called hybridization

Supercoiling results from strain on the molecule caused by under- or winding the double helix:

over-• Negatively supercoiled DNA is formed if the DNA is wound more loosely than in Watson-Crick DNA This form is required for most biologic reactions

• Positively supercoiled DNA is formed if the DNA is wound more tightly than in Watson-Crick DNA

Double-stranded DNA

Single-stranded DNA

Double-stranded DNA

Denaturation(heat)

Renaturation(cooling)

Figure I-1-9 Denaturation

and Renaturation of DNA

Figure I-1-9 Denaturation and

Renaturation of DNA

Chapter 1 ● Nucleic Acid Structure and Organization

• Topoisomerases are enzymes that can change the amount of

supercoil-ing in DNA molecules They make transient breaks in DNA strands by

alternately breaking and resealing the sugar-phosphate backbone For

example, in Escherichia coli, DNA gyrase (DNA topoisomerase II) can

introduce negative supercoiling into DNA

Nucleosomes and Chromatin

Expanded view of

a nucleosome

Figure I-1-10 Nucleosome and Nucleofilament

Structure in Eukaryotic DNAExpanded view

H2A

Figure I-1-10 Nucleosome and Nucleofilament

Structure in Eukaryotic DNA

Nuclear DNA in eukaryotes is found in chromatin associated with histones and

nonhistone proteins The basic packaging unit of chromatin is the nucleosome

• Histones are rich in lysine and arginine, which confer a positive charge

on the proteins

• Two copies each of histones H2A, H2B, H3, and H4 aggregate to form

the histone octamer

• DNA is wound around the outside of this octamer to form a

nucleo-some (a series of nucleonucleo-somes is nucleo-sometimes called “beads on a string”

but is more properly referred to as a 10nm chromatin fiber)

• Histone H1 is associated with the linker DNA found between

nucleo-somes to help package them into a solenoid-like structure, which is a

thick 30-nm fiber

• Further condensation occurs to eventually form the chromosome Each

eukaryotic chromosome in G0 or G1 contains one linear molecule of

double-stranded DNA

Cells in interphase contain 2 types of chromatin: euchromatin (more opened

and available for gene expression) and heterochromatin (much more highly

condensed and associated with areas of the chromosomes that are not

ex-pressed)

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DNA double helix 10 nm chromatin

(nucleosomes) 30 nm chromatin(nucleofilament) 30 nm fiber forms loops attachedto scaffolding proteins Higher order packaging

EuchromatinHeterochromatin

Figure I-1-12 An Interphase Nucleus

Nucleolus

Figure I-1-12 An Interphase Nucleus

During mitosis, all the DNA is highly condensed to allow separation of the sister chromatids This is the only time in the cell cycle when the chromosome structure is visible Chromosome abnormalities may be assessed on mitotic chromosomes by karyotype analysis (metaphase chromosomes) and by banding techniques (prophase or prometaphase), which identify aneuploidy, translocations, deletions, inversions, and duplications

Chapter 1 ● Nucleic Acid Structure and Organization

Review Questions

Select the ONE best answer.

1 A double-stranded RNA genome isolated from a virus in the stool of a child

with gastroenteritis was found to contain 15% uracil What is the

percent-age of guanine in this genome?

N

NN

3 Endonuclease activation and chromatin fragmentation are characteristic

features of eukaryotic cell death by apoptosis Which of the following

chro-mosome structures would most likely be degraded first in an apoptotic cell?

Behavioral Science/Social Sciences

4 A medical student working in a molecular biology laboratory is asked by her mentor to determine the base composition of an unlabeled nucleic acid sample left behind by a former research technologist The results of her analysis show 10% adenine, 40% cytosine, 30% thymine and 20% guanine

What is the most likely source of the nucleic acid in this sample?

Chapter 1 ● Nucleic Acid Structure and Organization

2 Answer: D A nucleoside consists of a base and a sugar The figure shows

the nucleoside adenosine, which is the base adenine attached to ribose

3 Answer: B The more “opened” the DNA, the more sensitive it is to

enzyme attack The 10-nm fiber, without the H1, is the most open

struc-ture listed The endonuclease would attack the region of unprotected

DNA between the nucleosomes

4 Answer: E A base compositional analysis that deviates from Chargaff’s

rules (%A = %T, %C = %G) is indicative of single-stranded, not

double-stranded, nucleic acid molecule All options listed except E are examples

of circular (choices A, B and C) or linear (choice D) DNA double helices

Only a few viruses (e.g parvovirus) have single-stranded DNA

DNA Replication and Repair

Learning Objectives

❏ Explain how DNA and RNA synthesis differ

❏ Know key steps in DNA replication

❏ Know major kinds of DNA repair

DNA REPLICATION

Genetic information is transmitted from parent to progeny by replication of

parental DNA, a process in which 2 daughter DNA molecules are produced that

are each identical to the parental DNA molecule During DNA replication, the

2  complementary strands of parental DNA are pulled apart Each parental

strand is then used as a template for the synthesis of a new complementary

strand (semiconservative replication) During cell division, each daughter cell

receives one of the 2 identical DNA molecules

2

Behavioral Science/Social Sciences

Replication of Prokaryotic and Eukaryotic Chromosomes

The process of DNA replication in prokaryotes and eukaryotes is compared below

Figure I-2-1 DNA Replication by a

Semi-Conservative, Bidirectional Mechanism

2 replication forks eventually meet, resulting in the production of 2 identical circular molecules of DNA

Each eukaryotic chromosome contains one linear molecule of dsDNA having multiple origins of replication Bidirectional replication occurs by means of a pair of replication forks produced at each origin Completion of the process results in the production of 2 identical linear molecules of dsDNA (sister chro-matids) DNA replication occurs in the nucleus during the S phase of the eukaryotic cell cycle The 2 identical sister chromatids are separated from each other when the cell divides during mitosis

Note

synthesize nucleic acids by forming phosphodiester (PDE) bonds

• Nucleases are enzymes that

hydro-lyze PDE bonds

– Exonucleases remove tides from the 5′ or the 3′ end of

nucleo-a nucleic nucleo-acid

– Endonucleases cut within the nucleic acid and release nucleic acid fragments

Chapter 2 ● DNA Replication and Repair

The structure of a representative eukaryotic chromosome during the cell cycle is

shown below

G2

SM

Panel A

Celldivision

Centromere

ds DNA

2 ds DNA(sister chromatids)

G1

Panel B

p

3211234q

Drawing of a replicated chromosome

Drawing of a stained replicated chromosome (metaphase)

Photograph of a stained replicated chromosome The individual chromatids and centromere are difficult to visualize in the photograph

Figure I-2-2 Panel A: Eukaryotic Chromosome Replication During S-Phase

Panel B: Different Representations of a Replicated Eukaryotic ChromosomePanel B: Different Representations of a Replicated Eukaryotic ChromosomeFigure I-2-2 Panel A: Eukaryotic Chromosome Replication During S-Phase

Behavioral Science/Social Sciences

COMPARISON OF DNA AND RNA SYNTHESIS

The overall process of DNA replication requires the synthesis of both DNA and RNA These 2 types of nucleic acids are synthesized by DNA polymerases and RNA polymerases, respectively. 

3' C-A-T-G-A-C-T-A-G-C-C-G-A-A-C-T-C-T-G-G-A 5'

A-C-U-G

RNA primer

Primer required for DNA synthesis (5'→3')using dNTP substrates

Primer not required for RNA synthesis (5'→3')using NTP substrates

A-C-U-G- A-T-C-G-G 3' 5'

High-fidelityDNA synthesis

A-C-U-G- A-T-C-G-G-C-T-T-G-A-G-A-C

5' A-U-C-G-G-U 3'

Mispaired nucleotidenot removed

Low-fidelityRNA synthesis

A-U-C-G-G-U-U-U-G-A-G-A-C

3' C-A-T-G-A-C-T-A-G-C-C-G-A-A-C-T-C-T-G-G-A 5' 3' C-A-T-G-A-C-T-A-G-C-C-G-A-A-C-T-C-T-G-G-A 5'

Table I-2-1 Comparison of DNA and RNA Polymerases

DNA Polymerase RNA Polymerase

Nucleic acid synthesized (5′→3′) DNA RNARequired template (copied 3′→5′) DNA* DNA*

Required substrates dATP, dGTP, dCTP, dTTP ATP, GTP, CTP, UTP

Proofreading activity (3′→5′ exonuclease) Yes No

* Certain DNA and RNA polymerases require RNA templates These enzymes are most commonly associated with viruses.

Chapter 2 ● DNA Replication and Repair

Similarities between DNA and RNA synthesis include:

• The newly synthesized strand is made in the 5′→3′ direction

• The template strand is scanned in the 3′→5′ direction

• The newly synthesized strand is complementary and antiparallel to the

template strand

• Each new nucleotide is added when the 3′ hydroxyl group of the

growing strand reacts with a nucleoside triphosphate, which is

base-paired with the template strand Pyrophosphate (PPi, the last two

phosphates) is released during this reaction

Differences include:

• The substrates for DNA synthesis are the dNTPs, whereas the

sub-strates for RNA synthesis are the NTPs

• DNA contains thymine, whereas RNA contains uracil

• DNA polymerases require a primer, whereas RNA polymerases do not

That is, DNA polymerases cannot initiate strand synthesis, whereas

RNA polymerases can

• DNA polymerases can correct mistakes (“proofreading”), whereas RNA

polymerases cannot. DNA polymerases have 3′ → 5′ exonuclease

activity for proofreading

STEPS OF DNA REPLICATION

The molecular mechanism of DNA replication is shown below The sequence of

events is as follows:

1 The base sequence at the origin of replication is recognized

2 Helicase breaks the hydrogen bonds holding the base pairs together This

allows the two parental strands of DNA to begin unwinding and forms 2

replication forks

3 Single-stranded DNA binding protein (SSB) binds to the single-stranded

portion of each DNA strand, preventing them from reassociating and

pro-tecting them from degradation by nucleases

4 Primase synthesizes a short (about 10 nucleotides) RNA primer in the

5′→3′ direction, beginning at the origin on each parental strand The

parental strand is used as a template for this process RNA primers are

re-quired because DNA polymerases are unable to initiate synthesis of DNA,

and can only extend a strand from the 3′ end of a preformed “primer.”

5 DNA polymerase III begins synthesizing DNA in the 5′→3′ direction,

be-ginning at the 3′ end of each RNA primer The newly synthesized strand is

complementary and antiparallel to the parental strand used as a template

This strand can be made continuously in one long piece and is known as

the “leading strand.”

• The “lagging strand” is synthesized discontinuously as a series of small

fragments (about 1,000 nucleotides long) known as Okazaki fragments

Each Okazaki fragment is initiated by the synthesis of an RNA primer

by primase, and then completed by the synthesis of DNA using DNA

polymerase III Each fragment is made in the 5′→3′ direction

Behavioral Science/Social Sciences

• There is a leading and a lagging strand for each of the two replication forks on the chromosome

6 RNA primers are removed by RNAase H in eukaryotes and an terized DNA polymerase fills in the gap with DNA In prokaryotes DNA polymerase I both removes the primer (5’ exonuclease) and synthesizes new DNA, beginning at the 3′ end of the neighboring Okazaki fragment

uncharac-7 Both eukaryotic and prokaryotic DNA polymerases have the ability to

“proofread” their work by means of a 3′→5′ exonuclease activity If DNA polymerase makes a mistake during DNA synthesis, the resulting unpaired base at the 3′ end of the growing strand is removed before synthesis continues

8 DNA ligase seals the “nicks” between Okazaki fragments, converting them

to a continuous strand of DNA

9 DNA gyrase (DNA topoisomerase II) provides a “swivel” in front of each replication fork As helicase unwinds the DNA at the replication forks, the DNA ahead of it becomes overwound and positive supercoils form DNA gyrase inserts negative supercoils by nicking both strands of DNA, pass-ing the DNA strands through the nick, and then resealing both strands

Quinolones are a family of drugs that block the action of ases Nalidixic acid kills bacteria by inhibiting DNA gyrase Inhibitors of eukaryotic topoisomerase II (etoposide, teniposide) are becoming useful as anticancer agents

topoisomer-The mechanism of replication in eukaryotes is believed to be very similar to this

However, the details have not yet been completely worked out The steps and proteins involved in DNA replication in prokaryotes are compared with those used in eukaryotes in Table I-2-2

Eukaryotic DNA Polymerases

• DNA α and δ work together to synthesize both the leading and lagging strands

• DNA polymerase γ replicates mitochondrial DNA

• DNA polymerases β and ε are thought to participate primarily in DNA repair DNA polymerase ε may substitute for DNA polymerase δ in certain cases

Telomerase

Telomeres are repetitive sequences at the ends of linear DNA molecules in karyotic chromosomes With each round of replication in most normal cells, the telomeres are shortened because DNA polymerase cannot complete synthe-sis of the 5′ end of each strand This contributes to the aging of cells, because eventually the telomeres become so short that the chromosomes cannot func-tion properly and the cells die

eu-Telomerase is an enzyme in eukaryotes used to maintain the telomeres It contains a short RNA template complementary to the DNA telomere sequence,

as well as telomerase reverse transcriptase activity (hTRT) Telomerase is thus able to replace telomere sequences that would otherwise be lost during replica-tion Normally telomerase activity is present only in embryonic cells, germ (reproductive) cells, and stem cells, but not in somatic cells

not present in adult somatic cells

• Inappropriately present in many cancer cells, contributing to their unlimited replication

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Chapter 2 ● DNA Replication and Repair

Cancer cells often have relatively high levels of telomerase, preventing the

telo-meres from becoming shortened and contributing to the immortality of

malig-nant cells

Table I-2-2 Steps and Proteins Involved in DNA Replication

Step in Replication Prokaryotic Cells

Eukaryotic Cells (Nuclei)

Origin of replication (ori) One ori site per

chromosome

Multiple ori sites per chromosomeUnwinding of DNA double

Single-stranded DNA-binding protein (SSB)

Synthesis of RNA primers Primase Primase

Removal of RNA primers DNA polymerase I

(5′→3′ exonuclease) RNase H (5′→3′ exonuclease)Replacement of RNA

with DNA

DNA polymerase I DNA polymerase δ

Joining of Okazaki

Reverse transcriptase is an RNA-dependent DNA polymerase that requires an

RNA template to direct the synthesis of new DNA Retroviruses, most notably

HIV, use this enzyme to replicate their RNA genomes DNA synthesis by reverse

transcriptase in retroviruses can be inhibited by AZT, ddC, and ddI

Eukaryotic cells also contain reverse transcriptase activity:

• Associated with telomerase (hTRT)

• Encoded by retrotransposons (residual viral genomes permanently

maintained in human DNA) that play a role in amplifying certain

repetitive sequences in DNA (see Chapter 7)

Bridge to Pharmacology

Quinolones and fluoroquinolones

inhibit DNA gyrase (prokaryotic

topoisomerase II), preventing DNA replication and transcription These drugs, which are most active against aerobic gram-negative bacteria, include:

• Levofloxacin

• Ciprofloxacin

• MoxifloxacinResistance to the drugs has developed over time; current uses include treatment

of gonorrhea and upper and lower urinary tract infections in both sexes

Bridge to Pharmacology

One chemotherapeutic treatment of HIV is the use of AZT (3′-azido-2′,3′-dideoxythymidine) or structurally related compounds Once AZT enters cells, it can be converted to the triphosphate derivative and used as a substrate for the viral reverse

transcriptase in synthesizing DNA from its RNA genome

The replacement of an azide instead of

a normal hydroxyl group at the 3′

position of the deoxyribose prevents further replication by effectively causing chain termination Although it is a DNA polymerase, reverse transcriptase lacks proofreading activity

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Behavioral Science/Social Sciences

Figure I-2-4 DNA Replication

Leading Strand Synthesis (Continuous)

1 Primase synthesizes the primer ( ) 5' to 3'.

2 DNA polymerases α and δ extend the primer, moving

into the replication fork (Leading strand synthesis)

3 Helicase ( ) continues to unwind the DNA.

Lagging Strand Synthesis (Discontinuous)

1 Primase synthesizes the primer ( ) 5' to 3'.

2 DNA polymerases α and δ extend the primer, moving

away from the replication fork (Lagging strand synthesis).

3 Synthesis stops when DNA polymerase encounters

the primer of the leading strand on the other side

of the diagram (not shown), or the primer of the previous (Okazaki) fragment

4 As helicase opens more of the replication fork, a

third Okazaki fragment will be added

RNase H (5' exoribonuclease activity) digests

the RNA primer from fragment 1 In the

eukaryotic cell, DNA polymerase extends the

next fragment (2), to fill in the gap

In prokaryotic cells DNA polymerase 1 has both

the 5' exonuclease activity to remove primers, and

the DNA polymerase activity to extend the next

fragment (2) to fill in the gap

In both types of cells DNA ligase connects

fragments 1 and 2 by making a phosphodiesterbond

This whole process repeats to remove all RNAprimers from both the leading and lagging strands

3'5'

5'3'

Origin

Helicase

3'

3'5'5'

+5'

5'

3'3'

Chapter 2 ● DNA Replication and Repair

DNA REPAIR

The structure of DNA can be damaged in a number of ways through exposure

to chemicals or radiation Incorrect bases can also be incorporated during

rep-lication Multiple repair systems have evolved, allowing cells to maintain the

sequence stability of their genomes If cells are allowed to replicate their DNA

using a damaged template, there is a high risk of introducing stable mutations

into the new DNA Thus any defect in DNA repair carries an increased risk of

cancer Most DNA repair occurs in the G1 phase of the eukaryotic cell cycle

Mismatch repair occurs in the G2 phase to correct replication errors

Table I-2-3 DNA Repair

Recognition/

Excision Enzyme

Repair Enzymes

Mismatched

base (G2)

DNA replication errors

A mutation on one of two genes, hMSH2 or hMLH1, initiates defective repair of DNA mismatches, resulting

in a condition known

as hereditary yposis colorectal cancer—HNPCC

nonpol-DNA polymeraseDNA ligase

DNA polymeraseDNA ligase

Repair of Thymine Dimers

Ultraviolet light induces the formation of dimers between adjacent thymines in

DNA (also occasionally between other adjacent pyrimidines) The formation of

thymine dimers interferes with DNA replication and normal gene expression

Thymine dimers are eliminated from DNA by a nucleotide excision-repair

mechanism

Bridge to PathologyDNA repair may not occur properly

when certain tumor suppressor genes

have been inactivated through mutation or deletion:

• The p53 gene encodes a protein

that prevents a cell with damaged DNA from entering the S phase

Inactivation or deletion associated with Li Fraumeni syndrome and many solid tumors

• ATM gene encodes a kinase sential for p53 activity ATM is

es-inactivated in ataxia telangiectasia, characterized by hypersensitivity

to x-rays and predisposition to

lym-phomas BRCA-1 (breast, prostate, and ovarian cancer) and BRCA-2

(breast cancer)

• The retinoblastoma Rb gene was

the first tumor suppressor gene cloned, and is a negative regulator

of the cell cycle through its ability

to bind the transcription factor E2F and repress transcription of genes required for S phase

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DNA ligasepolymerase

T

A TA3'

3'

5'

5'

Excision endonucleaseXeroderma pigmentosum (XP)

5'

5'5'

3'3'

3'3'

5'

5'3'

3'

T

A TA5'

5'3'

3'Nick

Figure I-2-5 Thymine Dimer Formation and Excision Repair

Steps in nucleotide excision repair:

• An excision endonuclease (excinuclease) makes nicks in the ester backbone of the damaged strand on both sides of the thymine dimer and removes the defective oligonucleotide

phosphodi-• DNA polymerase fills in the gap by synthesizing DNA in the 5′→3′

direction, using the undamaged strand as a template

• DNA ligase seals the nick in the repaired strand

Chapter 2 ● DNA Replication and Repair

Base excision repair: cytosine deamination

Cytosine deamination (loss of an amino group from cytosine) converts cytosine

to uracil The uracil is recognized and removed (base excision) by a uracil

gly-cosylase enzyme

• Subsequently this area is recognized by an AP endonuclease that

removes the damaged sequence from the DNA

• DNA polymerase fills in the gap

• DNA ligase seals the nick in the repaired strand

A summary of important genes involved in maintaining DNA fidelity and where

they function in the cell cycle is shown below

G2

S

M Mismatch

repair

• MSH2

• MLH1

Thymine dimer (bulky lesion) repair

• XP

• Nucleotide excision repair (cytosine deamination) Genes controlling entry into S-phase

• Rb

• p53

DNA polymerase proofreads during DNA synthesis

G1 G0

Figure I-2-6 Important Genes Associated with

Maintaining Fidelity of Replicating DNA

Figure I-2-6 Important Genes Associated with Maintaining Fidelity

of Replicating DNA

Diseases Associated with DNA Repair

Inherited mutations that result in defective DNA repair mechanisms are

associ-ated with a predisposition to the development of cancer

Xeroderma pigmentosum is an autosomal recessive disorder, characterized by

extreme sensitivity to sunlight, skin freckling and ulcerations, and skin cancer

The most common deficiency occurs in the excinuclease enzyme

Hereditary nonpolyposis colorectal cancer results from a deficiency in the

abil-ity to repair mismatched base pairs in DNA that are accidentally introduced

during replication

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Behavioral Science/Social Sciences

Hereditary nonpolyposis colorectal cancer (Lynch syndrome)

Hereditary nonpolyposis colorectal cancer (HNPCC) results from a mutation

in one of the genes (usually hMLH1 or hMSH2) encoding enzymes that carry

out DNA mismatch repair These enzymes detect and remove errors introduced into the DNA during replication In families with HNPCC, individuals may

inherit one nonfunctional, deleted copy of the hMLH1 gene or one tional, deleted copy of the hMSH2 gene After birth, a somatic mutation in the

nonfunc-other copy may occur, causing loss of the mismatch repair function This causes chromosomes to retain errors (mutations) in many other loci, some of which may contribute to cancer progression This is manifested in intestinal cells be-cause they are constantly undergoing cell division

One prominent type of error that accompanies DNA replication is lite instability In a patient with HNPCC, cells from the resected tumor show

microsatel-microsatellite instability, whereas normal cells from the individual (which still retain mismatch repair) do not show microsatellite instability Along with infor-mation from a family pedigree and histologic analysis, microsatellite instability may be used as a diagnostic tool

Note

Microsatellites (also known as short tandem repeats) are di-, tri-, and tetranucleotide repeats dispersed throughout the DNA, usually (but not exclusively) in noncoding regions

For example, TGTGTGTG may occur at a particular locus If cells lack mismatch repair, the replicated DNA will vary in the number of repeats at that locus, e.g., TGTGTGTGTGTG or TGTGTG This variation is microsatellite instability.

Xeroderma pigmentosum

Xeroderma pigmentosum is an autosomal recessive disorder (incidence 1/250,000) characterized by extreme sensitivity to sunlight, skin freckling, ulcerations, and skin cancer Carcinomas and melanomas appear early in life, and most patients die of cancer The most common deficiency occurs in the excision endonuclease

A 6-year-old child was brought to the clinic because his parents were concerned with excessive lesions and blistering in the facial and neck area

The parents noted that the lesions did not go away with typical ointments and creams and often became worse when the child was exposed to sunlight The physician noted excessive freckling throughout the child’s body, as well as slight stature and poor muscle tone

Xeroderma pigmentosum can be diagnosed by measurement of the relevant enzyme excision endonuclease in white cells of blood Patients with the disease should avoid exposure to any source of UV light

Chapter 2 ● DNA Replication and Repair

Review Questions

Select the ONE best answer.

1 It is now believed that a substantial proportion of the single nucleotide

substitutions causing human genetic disease are due to misincorporation

of bases during DNA replication Which proofreading activity is critical

in determining the accuracy of nuclear DNA replication and thus the

base substitution mutation rate in human chromosomes?

A 3′ to 5′ polymerase activity of DNA polymerase δ

B 3′ to 5′ exonuclease activity of DNA polymerase γ

C Primase activity of DNA polymerase α

D 5′ to 3′ polymerase activity of DNA polymerase III

E 3′ to 5′ exonuclease activity of DNA polymerase δ

2 The proliferation of cytotoxic T-cells is markedly impaired upon

infec-tion with a newly discovered human immunodeficiency virus, designated

HIV-V The defect has been traced to the expression of a viral-encoded

enzyme that inactivates a host-cell nuclear protein required for DNA

replication Which protein is a potential substrate for the viral enzyme?

A TATA-box binding protein (TBP)

B Cap binding protein (CBP)

C Catabolite activator protein (CAP)

D Acyl-carrier protein (ACP)

E Single-strand binding protein (SBP)

3 The deficiency of an excision endonuclease may produce an exquisite

sensitivity to ultraviolet radiation in xeroderma pigmentosum Which of

the following functions would be absent in a patient deficient in this

endonuclease?

A Removal of introns

B Removal of pyrimidine dimers

C Protection against DNA viruses

D Repair of mismatched bases during DNA replication

E Repair of mismatched bases during transcription

4 The anti-Pseudomonas action of norfloxacin is related to its ability to

inhibit chromosome duplication in rapidly dividing cells Which of the

following enzymes participates in bacterial DNA replication and is

directly inhibited by this antibiotic?

Behavioral Science/Social Sciences

5 Cytosine arabinoside (araC) is used as an effective chemotherapeutic agent for cancer, although resistance to this drug may eventually develop

In certain cases, resistance is related to an increase in the enzyme cytidine deaminase in the tumor cells This enzyme would inactivate araC to form

in DNA replication is most likely deficient in DKC patients?

A Synthesis of centromeres

B Synthesis of Okazaki fragments

C Synthesis of RNA primers

D Synthesis of telomeres

E Removal of RNA primers

7 Single-strand breaks in DNA comprise the single most frequent type of DNA damage These breaks are frequently due to reactive oxygen species damaging the deoxyribose residues of the sugar phosphate backbone

This type of break is repaired by a series of enzymes that reconstruct the sugar and ultimately reform the phosphodiester bonds between nucleotides Which class of enzyme catalyses the formation of the phos-phodiester bond in DNA repair?

Chapter 2 ● DNA Replication and Repair

Answers

1 Answer: E The 3′ to 5′ exonuclease activity of DNA pol δ represents the

proofreading activity of an enzyme required for the replication of human

chromosomal DNA DNA pol γ (mitochondrial) and DNA pol III

(pro-karyotic) do not participate in this process, short RNA primers are

replaced with DNA during replication, and new DNA strands are always

synthesized in the 5′ to 3′ direction

2 Answer: E TBP and CBP participate in eukaryotic gene transcription

and mRNA translation, respectively CAP regulates the expression of

prokaryotic lactose operons ACP is involved in fatty acid synthesis

3 Answer: B Nucleotide excision repair of thymine (pyrimidine) dimers is

deficient in XP patients

4 Answer: D Norfloxacin inhibits DNA gyrase (topoisomerase II).

5 Answer: D Deamination of cytosine would produce uracil.

6 Answer: D The enzyme is described as an RNA dependent DNA

poly-merase required for chromosome duplication in the nuclei of rapidly

dividing cells This enzyme is telomerase, a reverse transcriptase, that

replicates the ends (telomeres) of linear chromosomes

None of the other options have reverse transcriptase activity

7 Answer: C All DNA repair systems use a ligase to seal breaks in the sugar

phosphate backbone of DNA Although polymerase enzymes make

phos-phodiester bonds during DNA synthesis, these enzymes do not ligate

strands of DNA

Transcription and RNA Processing

Learning Objectives

❏ Use knowledge of types of RNA

❏ Understand concepts of prokaryotic messenger RNA

❏ Understand concepts of eukaryotic messenger RNA

❏ Demonstrate understanding of alternative splicing of eukaryotic

primary pre-mRNA transcripts

❏ Know key features of ribosomal RNA (rRNA)

❏ Know key features of transfer RNA (tRNA)

TRANSCRIPTION

The first stage in the expression of genetic information is transcription of the

information in the base sequence of a double-stranded DNA molecule to form

the base sequence of a single-stranded molecule of RNA For any particular

gene, only one strand of the DNA molecule (the template strand) is copied by

RNA polymerase as it synthesizes RNA in the 5′ to 3′ direction Because RNA

polymerase moves in the 3′ to 5′ direction along the template strand of DNA,

the RNA product is antiparallel and complementary to the template RNA

poly-merase recognizes start signals (promoters) and stop signals (terminators) for

each of the thousands of transcription units in the genome of an organism. 

The figure below illustrates the arrangement and direction of transcription for

several genes on a DNA molecule

Figure I-3-1 Transcription of Several Genes on a Chromosome

Figure I-3-1 Transcription of Several Genes on a Chromosome

3