DNA MetabolismReplication, Repair, Recombination

CH353

April 1, 2008

Challenges to DNA Replication

1. DNA strands with strong mutual binding affinity must be separated– DNA helicases use ATP for strand separation

2. DNA helix must be unwound for strand separation– Topoisomerases relax nearby overwound duplex DNA

3. Replication accuracy must overcome tautomer equilibrium (K ~10-4) – Error rates < 10-10 achieved by proofreading activities

4. Replication rates must match cell division rate and genome size– 2000 nucleotides / second required for bacterial genome

5. Mechanism required for bidirectional replication of both strands– Semidiscontinuous replication of leading and lagging strands

6. Replicating linear DNA requires special structures and enzymes– Telomerase adds telomeres to ends for primed 5’→3’ synthesis

Basic Properties of DNA Replication

• Replication is semiconservative– Each strand is template for synthesis

of new strand

• Replication begins at an origin– Replication propagates from origin in

replication forks, where original DNA is unwound and new strands made

• Replication is semidiscontinuous– DNA synthesized in 3’→5’ direction

– Continuous synthesis of leading strand (direction same as replication)

– Discontinuous synthesis of lagging strand (opposite to replication)

Enzymes Involved in Replication

• Polymerases (classified by template and product)– DNA-dependent DNA synthesis (DNA polymerase)– DNA-dependent RNA synthesis (primase)– RNA-dependent DNA synthesis (telomerase)

• Nucleases (classified by substrate and action)– deoxyribonucleases (DNases); ribonucleases (RNases)– endonucleases; exonucleases (5’, 3’ or both)

• Ligases – join ends of DNA (or RNA) with phosphodiester bonds• Helicases – unwinds duplex DNA (requires ATP)• Topisomerases – interconvert topoisomers

– Topoisomerase I – relaxes supercoils– Topoisomerase II – forms negative supercoils (requires ATP)

DNA Synthesis Reaction

General reaction: (dNMP)n + dNTP → (dNMP)n+1 + PPi

Template : Primer + n dNTP → Template : Primer-(dNMP)n + n PPi

• Requires DNA template and DNA or RNA primer (with 3’-OH)

• Requires complementary dNTP’s and Mg2+

• Reaction driven by hydrolysis of PPi; ∆G’º = -19 kJ/mol

Mechanism: nucleophilic attack by 3’-OH on α-phosphate of dNTP with PPi as leaving group

DNA Polymerases of E. coli

• DNA polymerases VI and V are involved in DNA repair (TLS polymerases)– no proofreading activity; base specificity 102-fold lower; error rate ~10-3

Proofreading Activity of DNA Polymerases

• The 3’→5’ exonuclease activity of DNA polymerases provides error correction capability for higher fidelity of DNA replication

• Proofreading activity is hydrolytic; not reverse of polymerase activity• improves accuracy ~102 to 103 fold (net error rate: 10-6 to 10-8)

Nick Translation

• 5’→3’ exonuclease activity of DNA polymerase I removes nucleotides (DNA or RNA) from 5’ end of nick

• DNA polymerase activity adds nucleotides to 3’ end of nick

• Nick is “translated”, i.e. moves along DNA strand

• 5’→3’ exonuclease activity is on terminal domain of polymerase I

• Klenow fragment (Mr 68,000) of DNA polymerase I has polymerase and proofreading activities, but no 5’→3’ exonuclease activity

E. coli DNA Polymerase III (Replisome)

Components of Replisome:

• 2 core polymerases ()2

• 1 clamp loading () complex (2’)

• 1 DNA helicase (DnaB6)

joined together by 2 subunits

• 2 sliding clamps (2)2

Replication of the E. coli Chromosome

Stage 1: Initiation1. Complex of 4-5 DnaA subunits binds to 4

9-bp repeats in replication origin (oriC)

2. DnaA complex recognizes and denatures DNA in adjacent 13-bp repeats (A-T rich); requires histone-like protein (HU) and ATP

3. DnaC loads DnaB helicase unto unwound DNA; DnaB forms a hexameric ring around each DNA strand

4. DNA primase binds to DnaB helicase and synthesizes RNA primers on both strands

Replication of E. coli Chromosome

Stage 2: Elongation• DNA gyrase relaxes supercoils;

DNA helicase unwinds DNA ahead of replication fork

• SSB binds single stranded DNA

• Leading strand synthesis: polymerase III extends DNA from RNA primer at origin

• DNA primase synthesizes RNA primers on opposite strand

• Lagging strand synthesis: polymerase III synthesizes DNA from other RNA primers

Coordinating the Replication of Both Strands

from Pomerantz & O’Donnell (2007) Trends Microbiol. 15, 156

Final Step in Lagging Strand Synthesis

For each segment of lagging strand (Okazaki fragment)

• DNA polymerase I removes RNA primer and replaces it with new DNA (nick translation)

• DNA ligase closes nick in DNA– needs ATP or NAD+ as AMP donor

– needs 5’ phosphate and 3’ OH

– activates 5’ phosphate with AMP

– nucleophilic attack by 3’OH; AMP is leaving group

Replication of the E. coli Chromosome

Stage 3: Termination• Clusters of 20 bp Ter sequences trap

replication forks from leaving locus• Ter binding sites for Tus (terminus

utilization substance) proteins• Meeting of replication forks creates

concatenated chromosomes that are resolved with DNA topoisomerase IV

Replication and Chromosomal Partitioning

• 2 replisomes are attached to bacterial inner membrane (one for each replication fork)

• original DNA is fed through 2 replisomes; replicated DNA is sent in opposite directions

Eukaryotic DNA Replication Systems

Analogous to bacterial DNA replication systems• OriC Autonomously replicating sequences (ARS) • DnaA Origin recognition complex (ORC) binds to ARS• DnaC CDC6 and CDT1 mediate helicase loading• DnaB MCM2 – MCM7 form hexameric helicase ring• DnaG DNA polymerase primase activity• SSB RPA (replication protein A) single-stranded binding

DNA Polymerase III • [core] DNA polymerase • [complex] RFC (replication factor C) clamp loading• [ clamp] PCNA (proliferating cell nuclear antigen) clamp• DNA Pol I DNA polymerase

Problem of Replicating Linear Genomes

• DNA synthesis requires a primer with 3’-OH; terminal primer of a genome must be RNA, since RNA synthesis is primer independent

• After hydrolyzing the terminal RNA primer there must be a way for replicating the 3’ ends; or else the genome would become shorter with each cycle of replication

• Circular genomes avoid the problem, i.e. there is always a DNA primer upstream of the replaced RNA primer

• Some linear viral and bacteriaphage genomes have terminal repeats for replacing lost terminal sequences

• Most linear eukaryotic genomes have telomeres at the ends of each chromosome, which become shortened after each replication cycle

• Sustained replication requires a special mechanism for replacing the lost telomere sequences

Synthesis of Telomeres

• Telomerase adds telomeres to 3’ termini of linear genomic DNA (chromosomes)

• Internal template RNA hybridizes to DNA at -TTTTG-3’ sequence

• Telomerase adds –GGGTTTTG-3’ to end of DNA

• Telomerase translocates to added DNA and additional sequences are added

• Telomerase is not expressed in all cells• Undifferentiated stem cells and tumor

cells produce telomerase (cancer target)• Terminal T-loops protect telomeres from

nucleases and DNA repair enzymes

DNA Repair Systems

• Mismatch repair (mismatches)

– binds to mismatch, excises flanking DNA, synthesizes new DNA

• Base-excision repair (defective bases)

– removes base, removes AP nucleotide, adds new nucleotide(s)

• Nucleotide-excision repair (bulky DNA lesions)

– excises oligonucleotide with lesion, synthesizes new DNA

• Direct repair (pyrimidine dimers, methylated bases)

– enzymatic reversal of modification to base on DNA

• Error-prone translesion DNA synthesis (unrepaired lesion)

– SOS response, low fidelity DNA synthesis

• Recombinational DNA repair (unrepaired lesion or break)

Mismatch Repair

Base-Excision Repair

Nucleotide-Excision Repair

Direct Repair

Enzymes that modify bases attached to DNA• DNA photolyases

– enzyme uses light energy to reverse pyrimidine dimers– not present in placental mammals

• O6-methylguanine DNA methyltransferase– “enzyme” accepts methyl group and is permanently inactivated

• AlkB– oxidative demethylation of 1-methyladenine and 3-methylcytosine– α-ketoglutarate-Fe2+-dependent dioxygenase enzyme

Repair at Stalled Replication Forks

DNA Recombination

• Homologous genetic (general) recombination– genetic exchange between 2 DNAs sharing regions of nearly

identical sequence– Occurs most frequently during meiosis (crossing over)

• Site-specific recombination– Genetic exchange occuring at particular DNA sequences– Requires specific recombinase (integrase) and recombination

sites on each DNA– Cre-lox system used for conditional knockout mutations

• DNA transposition – “jumping genes”

– Genetic exchange involves transposable elements (transposons)– Rearrangement of immunoglobulin genes

Crossing Over during Meiosis

Recombination during Meiosis

Recombinational DNA Repair

Replication forks stalled at:

• DNA lesion– fork regression – replication with DNA Pol I

• DNA nick– strand invasion – Holliday junction resolution

Replication resumes• Origin-independent replication

restart with PriA, B and C, and DnaB, C, G and T