DNA Metabolism Replication, Repair, Recombination

1. DNA MetabolismReplication, Repair, Recombination CH353 April 1, 2008

2. Challenges to DNA Replication • DNA strands with strong mutual binding affinity must be separated • DNA helicases use ATP for strand separation • DNA helix must be unwound for strand separation • Topoisomerases relax nearby overwound duplex DNA • Replication accuracy must overcome tautomer equilibrium (K ~10-4) • Error rates < 10-10 achieved by proofreading activities • Replication rates must match cell division rate and genome size • 2000 nucleotides / second required for bacterial genome • Mechanism required for bidirectional replication of both strands • Semidiscontinuous replication of leading and lagging strands • Replicating linear DNA requires special structures and enzymes • Telomerase adds telomeres to ends for primed 5’→3’ synthesis

3. 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)

4. 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)

5. 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 Synthesis Reaction

6. 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

7. 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)

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

9. E. coli DNA Polymerase III (Replisome) Components of Replisome: • 2 core polymerases (aeq)2 • 1 clamp loading (g) complex (t2gdd’) • 1 DNA helicase (DnaB6) joined together by 2 t subunits • 2 sliding clamps (b2)2

10. Replication of the E. coli Chromosome Stage 1: Initiation • Complex of 4-5 DnaA subunits binds to 4 9-bp repeats in replication origin (oriC) • DnaA complex recognizes and denatures DNA in adjacent 13-bp repeats (A-T rich); requires histone-like protein (HU) and ATP • DnaC loads DnaB helicase unto unwound DNA; DnaB forms a hexameric ring around each DNA strand • DNA primase binds to DnaB helicase and synthesizes RNA primers on both strands

11. 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

12. Coordinating the Replication of Both Strands

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

14. 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

15. 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

16. 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

17. 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 a primase activity • SSB RPA (replication protein A) single-stranded binding DNA Polymerase III • [core] DNA polymerase d • [g complex] RFC (replication factor C) clamp loading • [b clamp] PCNA (proliferating cell nuclear antigen) clamp • DNA Pol I DNA polymerase e

18. 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

19. 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

20. 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)

21. Mismatch Repair

22. Base-Excision Repair

23. Nucleotide-Excision Repair

24. 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

25. Repair at Stalled Replication Forks

26. 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

27. Crossing Over during Meiosis

28. Recombination during Meiosis

29. 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