Lecture Connections 25 | DNA Metabolism

What is DNA Metabolism?• While functioning as a stable storage of genetic information, the structure of DNA is far from static: – A new copy of DNA is synthesized with high fidelity bef

Lecture Connections

25 | DNA Metabolism

© 2009 W H Freeman and Company

What is DNA Metabolism?

• While functioning as a stable storage of genetic

information, the structure of DNA is far from static:

– A new copy of DNA is synthesized with high fidelity before each cell division

– Errors that arise during or after DNA synthesis are constantly checked for, and repairs are made

– Segments of DNA are rearranged either within a chromosome

or between two DNA molecules giving offspring a novel DNA

• DNA metabolism consists of a set of enzyme catalyzed and tightly regulated processes that achieve these tasks

The Substrate that Encodes its

Own Metabolisms

The Meselson-Stahl Experiment

• The Meselson-Stahl experiment was about the origin of the two strands in each of the daughter genomes

• Cells were grown on a medium containing only

15N isotope until all their DNA became fully 15N labeled

• Cells were then switched to 14N medium and

allowed to divide once

• CsCl density gradient centrifugation was used to determine the mass of genomic DNA before and after each round of replication

DNA Replication is Semiconservative

• The Meselson-Stahl experiment showed that the

nitrogen used for the synthesis of new dsDNA becomes equally divided between the two

daughter genomes

• This suggests a semiconservative replication

mechanism

Replication of Circular DNA is

Bidirectional

• Both strands are replicated simultaneously

Synthesis of the Leading and

Lagging Strands

• Synthesis always occurs by addition of new

nucleotides to the 3’ end

• The leading strand is made continuously as the replication fork advances

• The lagging strand is made discontinuously in short pieces (Okazaki fragments) that are then joined

DNA Elongation Chemistry

• Parental DNA strand serves as a template

• Nucleotide triphosphates serve as substrates in strand synthesis

• Hydroxyl at the 3’ end of growing chain makes a bond to the -phosphorus of nucleotide

• Pyrophosphate is a good leaving group

DNA Synthesis is Catalyzed by

DNA Polymerases

• Mg++ on the right coordinates to the -phosphate and stabilize the negatively charged transition

state

Correct Geometry of Base Pairs

allows High Fidelity

Errors During the Synthesis are

Activity (1)

Errors During the Synthesis are

Activity (2)

Three DNA Polymerases in E coli

• Polymerase I is most abundant but its

primary function is in clean-up during

replication, repair, and recombination

• Polymerase II is probably responsible for

DNA repair

• Polymerase III is responsible for DNA

replication

Structure of Bacterial DNA Polymerases is Well Understood

• Structure of the Klenow fragment of DNA

polymerase I

• Structure of DNA Polymerase III bound to DNA

Synthesis of Okazaki Fragments

Synthesis of Leading and Lagging

Strands

DNA Ligase Seals the Nicks in

the Lagging Strand

DNA Repair and Mutations

• Chemical reactions and some physical processes

constantly damage genomic DNA

– At the molecular level, damage usually involves changes in the structure of one of the strands

– Vast majority are corrected by repair systems using the other

Types of DNA Repair Systems

• Mismatches arise from occasional incorporation

of incorrect nucleotides

• Abnormal bases arise from spontaneous

deamination reactions or via chemical alkylation

• Pyrimidine dimers form when DNA is exposed

to UV light

• Backbone lesions occur from exposure to

ionizing radiation

Methylation of DNA in Bacteria

• E coli DNA is fully methylated before replication

at GATC sites

• The newly synthesized strand is unmethylated for

a short period after synthesis

• The newly synthesized strand becomes

methylated before the cells divide

• Methylation is thought to ensure that cells do not divide before replication is complete

Methylation of DNA and Repair (1)

• The newly synthesized strand is unmethylated

for a short period after synthesis

• Any replication errors must reside in the

unmethylated strand

• Methyl-directed mismatch repair system will

cleave the unmethylated strand in the initial

part of the repair process

Methylation of DNA and Repair (2)

• The cleaved strand with incorrect nucleotide is degraded by exonucleases and rebuilt by DNA polymerase

Base Excision Repair

DNA Recombination

– within a chromosome

– from one chromosome to another

• Such recombination is involved in many biological

processes

– Repair of DNA

– Segregation of chromosomes during meiosis

– Enhancement of generic diversity

• In sexually reproducing organism, recombination and mutations are two driving forces of evolution

• Recombination of co-infecting viral genomes may

enhance virulence and provide resistance to antivirals

Homologous Recombination

• Genetic exchange occurs between two molecules that share an extended region of nearly identical sequences

Holliday Intermediate Between

Two Bacterial Plasmids

Chapter 25: Summary

• DNA replication is catalyzed by DNA polymerases that use one of the strand as a template while adding new nucleotides

to the 3’-end of the newly synthesized chain

• Several mechanisms exist to correct mismatches and other changes in DNA

• During DNA repair, the information encoded in the parent

strand can be used to make corrections in the daughter