Chapt 20 DNA Replication I: Basic Mechanism and Enyzmology

1. Chapt 20 DNA Replication I:Basic Mechanism and Enyzmology Student learning outcomes: • Describe general features of semi-conservative DNA replication: leading, lagging, strands; requirement for primers; bidirectional, rolling circle • Describe DNA polymerases: general enzymology and comparison of prokaryotes, eukaryotes • Describe major types of DNA damage and repair: Important Figures: 1, 4, 7*, 9, 10, 13, 14, 15, 16, 18, 22, 24, 26, 27, 28, 29, 30, 31*, 32, 33*, 34, 36, 37, 38, 39, 40, 41 Review problems: 8-11, 13-15, 22-24, 27-29, 32-35; AQ4

2. 20.1 General Features of DNA Replication • Double helical model for DNA: complementary strands • Each strand is template for new partner strand • Semiconservative model for DNA replication 5’ -> 3’ • Leading strand continuous synthesis • Half-discontinuous (short pieces on lagging strand are later stitched together) • Requires RNA primers • Usually bidirectional (bacterial and eukaryotes) • Origin of replication (ori): fixed starting point • Replicon: DNA under control of one ori

3. Semi-discontinuous Replication • DNA polymerase only synthesizes 5’3’ direction • RNA primers 10-12 nt long in E. coli • Leading strand replicates continuously in direction of movement of fork • Lagging strand replicates discontinuously in direction opposite to fork (as 1-2 kb Okazaki fragments) Fig. 7

4. Bidirectional Replication • Replication structure resembles Greek letter,  • DNA replication begins with creation of “bubble” –small region where parental strands separated, progeny DNA synthesized • As bubble expands, replicating DNA is  shape Fig. 9

5. Rolling Circle Replication • Circular DNAs can replicate as rolling circle • One strand of dsDNA is nicked, 3’-end extended (leading) • Uses intact DNA strand as template • 5’-end gets displaced; lagging synthesis fills in • Phage l:leading strand elongates continuously; • displaced strand serves as template for discontinuous, lagging strand synthesis; can get many genome sized piece Fig. 20

6. 20.2 Enzymology of DNA Replication • >30 different polypeptides to replicate E. coli DNA • Biochemical purifications, conditional mutants to examine activities of proteins (essential activities) • DNA polymerases – enzymes that make DNA • Require primer, Mg++, buffer, dNTPs • 3 DNA polymerases in E. coli: • pol I - repair enzyme • pol II - non-essential • pol III – the real replication enzyme

7. DNA Polymerase I Fig. 16 • E. coli DNA polymerase I – • first enzyme (1958, Arthur Kornberg) • Pol I has 3 distinct activities: • DNA polymerase • 3’5’ exonuclease – proofreading • 5’3’ exonuclease – degrades strand ahead of it • Can remove primers • Mild proteolytic treatment ->2 polypeptides • Klenow fragment (lacks 5’->3’ exonuclease) • Can fill in sticky ends left by restriction enzymes (Fig. 15) • Steitz structure 1987

8. Pol III Holoenzyme Pol III core has 3 subunits: catalysis, proofread, Pol III g complex has 5 subunits – DNA-dependent ATPase Pol III holoenzyme includes b subunit Charles McHenry (CU SOM) biochemical studies

9. Eukaryotes have multiple DNA Polymerases Mammalian cells 5 different DNA polymerases • Polymerases d and a replicate both DNA strands • PCNA factor helps with processivity

10. Other enzymes for replication E. coli • Helicase – uses ATP to unwind strands • Creates positive supercoils • dnaB gene • Single strand DNA binding protein – SSB • Stimulates polymerization • DNA gyrase (topoisomerase II) • Negative supercoils, swivel

11. 20.3 DNA Damage and Repair • DNA can be damaged in many different ways: • Cells have many ways to repair damage, easier to repair before DNA is replicated • if unrepaired, damage can lead to mutation • DNA damage is not the same as mutation, but it can lead to mutation • DNA damage is chemical alteration • Mutation is inherited change in base pair • Common examples of DNA damage • Base modifications caused by alkylating agents • Pyrimidine dimers caused by UV radiation

12. Alkylation of Bases Causes Damage • Alkylation - process where electrophiles: • Attack negative centers • Add carbon-containing groups (alkyl groups) Common targets (red): N7 of G, N3 of A, phosphodiester bond O6 of G Fig. 27

13. Alkylation of Bases Causes Damage Alkylating agents like ethylmethane sulfonate (EMS): • Some alkylations don’t change base-pairing – innocuous • Others cause DNA replication to stall: • Cytotoxic • Mutations if cell attempts to replicate without repair • Others change base-pairing properties, so are mutagenic: • Ethyl O6-G mispairs with T -> GC ->AT transition mutation Fig. 28

14. UV Radiation Damages DNA • Ultraviolet rays (260 nm) • Comparatively low energy • Moderate type of damage • Result in formation of pyrimidine dimers • Mostly T-T dimers • T-T dimers distort DNA, block replication and transcription Fig. 29: Thymine dimers have cyclobutane ring

15. Ionizing Radiation Damages DNA • Gamma and x-rays • Much more energetic • Ionize molecules around DNA • Highly reactive free radicals attack DNA • Alter bases • Break DNA strands • Especially double strand • (useful cancer therapy) C8-> Fig. 30: oxidative damage forms 8-oxo-guanine; At replication, A often is inserted opposite -> mutation

16. UV DNA Damage can be directly reversed • Photoreactivation (light repair) • DNA photolyase uses energy from near-UV to blue light to break bonds holding 2 pyrimidines together • Enzyme in most organisms (not placental mammals) Fig. 31

17. Reversing High Energy DNA Damage • O6 alkylations on G residues directly reversed by enzyme O6-methylguanine methyltransferase • Enzyme accepts alkyl group onto SH of Cys - and is inactivated (suicide enzyme) • In E. coli, enzyme is induced by DNA alkylation Fig. 32

18. Excision Repair • Only small percentage of DNA damage products are directly reversed • Excision repair removes most damaged nucleotides: • Damaged DNA is removed • Replaced with fresh DNA • Both base and nucleotide excision repair

19. Base Excision Repair (BER): specific enzymes remove damaged base DNA glycosylase • Extrudes base in damaged base pair, and clips out • Leaves apurinic or apyrimidinic (AP) site that attracts DNA repair enzymes DNA repair enzymes • Remove remaining deoxyribose phosphate • Replace with normal nucleotide, ligate Fig. 33

20. Eukaryotic BER Fig. 34 • DNA polymerase b fills in missing nucleotide Makes mistakes, not proofread • APE1 proofreads • (AP endonuclease) • Repair of 8-oxoG sites is special case of BER: • After replication, A often is inserted; A can be removed by specialized adenine DNA glycosylase • Before replication, oxoG paired with C; the oxoG is removed by oxoG DNA glycoslyase (hOGG1)

21. Nucleotide Excision Repair (NER) • NER handles bulky damage that distorts DNA • Including Thymine dimers, large adducts • Specific endonucleases clip DNA strand on either side of lesion, remove single strand, resynthesize and rejoin. • Xeroderma pigmentosum (XP) people have hereditary increased skin cancer; lack NER enzymes

22. NER in E. coli • Excinuclease (UvrABC) cuts either side of damage • Remove 12-13 nt oligonucleotide • Pol I fills in using top strand as template • DNA ligase seals nick Fig. 36

23. Eukaryotic NER uses 2 paths • GG-NER • Complex of XPC and hHR23B initiates repair, binds lesion • limited DNA melting • XPA and RPA recruited • TFIIH joins, helicase expands melted region • RPA binds 2 excinucleases (XPF, XPG) cleaves • Releases damage 24-32 nt • Transcription-Coupled (TC-NER) resembles GG-NER Except: • RNA polymerase plays role of XPC in damage sensing and initial DNA melting

24. Human Global Genome NER • Complex of XPC and hHR23B initiates repair, binds lesion • Limited DNA melting • XPA + RPA recruited • TFIIH joins, helicase expands melted region • RPA binds 2 excinucleases (XPF, XPG), • Cleaves, releases damage 24-32 nt Fig. 37

25. Transcription-Coupled (TC)-NER • Resembles GG-NER: • RNA polymerase plays role of XPC in damage sensing and initial DNA melting • RNAP stalls Fig. 37

26. Double-Strand Break (DSB) Repair in Eukaryotes • dsDNA breaks in eukaryotes are very dangerous • Broken chromosomes • If not repaired, lead to cell death • In vertebrates, also leads to cancer • Eukaryotes deal with dsDNA breaks in 2 ways: • Homologous recombination with good chromsome • Nonhomologous end-joining (NHEJ) has errors • Chromatin remodeling has role in dsDNA break repair] [

27. Model for Nonhomologous End-Joining • Ku and DNA-PKcs bind at DNA ends and let ends find microhomology • 2 DNA-PK complexes phosphorylate each other: • Catalytic subunit dissociates • DNA helicase activity of Ku unwinds DNA ends • Extra flaps of DNA removed, gaps filled, ends ligated • Inaccurate process, DNA is lost Fig. 38

28. Mismatch Repair • Recognizes parental DNAby its methylated A in GATC sequence (E. coli) • Corrects mismatch in progeny strand • Eukaryotes use part of repair system – unclear how distinguish strands at mismatch • HNPCC colon cancer- defects in repair of mismatch damage cause instability of microsatellite regions, many mutations Fig. 39

29. Coping with DNA Damage Without Repairing It • Direct reversal and excision repair are true repair processes - accurate • Eliminate defective DNA entirely • Cells also copes with damage by skirting it • Not true repair mechanism • Damage bypass mechanism: • gives time to repair • cell can replicate, fix damage later

30. Recombination Repair • Gapped DNA strand across from damaged strand recombines with normal strand in other daughter DNA duplex after replication • Must occur before segregation • Solves gap problem • Leaves original damage unrepaired – fix later Fig. 40

31. Error-Prone Bypass (SOS) • Induce SOS response • Activates recA protease • UmuC/D dimer is DNA pol V • Causes DNA to replicate even though damaged region not read correctly • Errors in newly made DNA, but cell lives • Mutants of umu genes die, but do not have mutations Fig. 41 Recall, UV damage to cell can induce SOS path, which causes cleavage of lambda repressor, return of prophage to lytic cycle

32. Error-Prone Bypass in Humans • Humans have relatively error-free bypass system that inserts dAMPs across from pyrimidine dimer • Specialized DNA polymerases are activated • Replicate thymine dimers correctly • Uses DNA polymerase  plus another enzyme to replicate a few bases beyond lesion • Polymerase is not really error-free • If DNA polymerase  gene is defective, DNA polymerase  and others take over, more errors

33. Review questions • 8. Diagram rolling circle replication of lambda • 12, 16. List the different DNA polymerases in E. coli and eukaryotes and explain their roles. • 24. Compare/ contrast base excision repair and nucleotide excision. • 33. Diagram recombination repair in E.coli.