Chen Yonggang Zhejiang Univ. School of Medicine Research Building C-616 Chenyg9@163

1. A DNA Repair Overview Chen Yonggang Zhejiang Univ. School of Medicine Research Building C-616 Chenyg9@163.com

2. Excellent Review Articles • Friedberg, EC (2003) DNA damage and repair.Nature 421:436-440. • Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S (2004) Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints.Annu Rev Biochem 73: 39-85.

3. Importance of Repair • DNA is the only biological macromolecule that is repaired. All others are replaced. • More than 100 genes are required for DNA repair, even in organisms with very small genomes. • Cancer is a consequence of inadequate DNA repair.

4. DNA can be damaged by a variety of processes 1, Some spontaneously: deamination C U 2, Others catalyzed by enviromental agents • UV light: dimer • Chemicals: (1) deaminating agents (2) alkylating agents c) Oxidative damage: hydrogen peroxide, hydroxyl radicals, superoxide radicals

5. Spontaneous base loss: Several thousand purines and serval thousand pyrimidines per haploid genome per day! AP Site: apyrimidinic site or apurinic site

6. Spontaneous deamination: ~100 uracils per haploid genome per day. Also: Adenine to hypoxanthine Guanine to xanthine 5-methyl cytosine to thymine

7. “Reactive Oxygen Species” (ROS) include O•, O-O•, HOOH, •OH Thymine Thymine Glycol

8. Spontaneous production of 3-Methyl Adenine by S-Adenosylmethionine: Several hundred per haploid genome per day!

9. Effects of Sunlight: (photodamage) Cyclobutane pyrimidine dimers (CPDs) T-T>T-C, C-T>C-C DNA helix bends 7-9°

10. Effects of Sunlight: (photodamage) Pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) T-C>C-C>T-T>C-T DNA helix bends 44°

11. Some Additional Types of Damage: • Replication errors • Intra- and inter-strand crosslinks • DNA-protein crosslinks • Strand breaks

12. Types of DNA Repair • Direct repair(Direct reversal of damage) • Excision repair(Excision of damaged region, followed by precise replacement) • Recombination repair(strand break repair) • Damage bypass

13. An Example of Direct Repair: “Photoreactivation” MTHF or 8-HDF FADH-

14. Additional Examples of Direct Repair • 6-4 photolyases • Ligation of nicks

15. Excision Repair Takes advantage of the double-stranded (double information) nature of the DNA molecule. • Mismatch repair • Base excision repair • Nucleotide excision repair

16. Mismatch repair in E. coli • Excision by UvrD (Helicase II and single-strand exonuclease) • Gap filling by Polymerase I(in E. coli); Ligation by DNA ligase

19. Base Excision Repair

20. Base Excision Repair • Several variations, depending on nature of damage, nature of glycosylase, and nature of DNA polymerase. • All have in common the following steps: • Removal of the incorrect base by an appropriate DNA N-glycosylase to create an AP site. • An AP endonuclease nicks on the 5’ side of the AP site to generate a 3’-OH terminus. • Extension of the 3’-OH terminus by a DNA polymerase.

21. An example of a DNA N-glycosylase: Pinch-push-pull mechanism suggested by crystal structures of glycosylases.

22. Some DNA N-glycosylases have AP lyase activity.

23. Initial steps of base-excision repair

24. Final steps of base-excision repair (DNA polymerase β pathway; short patch repair

25. Final steps of base-excision repair (replication pathway)

26. Nucleotide Excision Repair • Extremely flexible • Corrects any damage that distorts the DNA molecule • In all organisms, NER involves the following steps: • Damage recognition • Binding of a multi-protein complex at the damaged site • Double incision of the damaged strand several nucleotides away from the damaged site, on both the 5’ and 3’ sides • Removal of the damage-containing oligonucleotide from between the two nicks • Filling in of the resulting gap by a DNA polymerase • Ligation

27. S. cerevisiae protein Human protein Probable function Rad4 XPC GGR (also required for TC-NER in yeast); works with HR23B; binds damaged DNA; recruits other NER proteins Rad23 HR23B GGR; cooperates with XPC (see above); contains ubiquitin domain; interacts with proteasome and XPC Rad14 XPA Binds and stabilizes open complex; checks for damage Rpa1,2,3 RPAp70,p32,p14 Stabilizes open complex (with Rad14/XPA) Ssl2 (Rad25) XPB 3' to 5' helicase Tfb1 GTF2H1 ? Tfb2 GTF2H4 ? Ssl1 GTF2H2 Zn finger; DNA binding? Tfb4 GTF2H3 Ring finger; DNA binding? Tfb5 TFB5; TTD-A Stabilization of TFIIH Rad3 XPD 5' to 3' helicase Tfb3/Rig2 MAT1 CDK assembly factor Kin28 Cdk7 CDK; C-terminal domain kinase; CAK Ccl1 CycH Cyclin Rad2 XPG Endonuclease (3' incision); stabilizes full open complex Rad1 XPF Part of endonuclease (5' incision) Rad10 ERCC1 Part of endonuclease (5' incision) Proteins Required for Eukaryotic Nucleotide Excision Repair

28. Nucleotide Excision Repair

29. Early Stages of Global Genome Repair

30. Initial Steps of Transcription-Coupled NER

31. Final Steps of Eukaryotic NER

32. Some of the proteins required for eukaryotic NER S. Cerevisiae Human Protein Probable function Rad 4 XPC GGR (also required for TC-NER in yeast; works with HR23B; binds damaged DNA; recruits other NER proteins Rad 23 HR23B GGR: cooperates with XPC; contains ubiquitin domain; interacts with proteasome and XPC Rad 14 XPA Binds and stabilizes open complex; checks for damage Rpa1, 2, 3 RPA p70, p32, p14 Stabilizes open complex (with Rad14/XPA) Ssl2 (Rad25) XPB 3’ to 5’ helicase Tfb1 GTF2H1 ? Tfb2 GTF2H4 ? Ssl1 GTF2H2 Zn Finger; DNA binding? Tfb4 GTF2H3 Ring Finger; DNA binding? Tfb5 TFB5; TTD-A Stabilization of TFIIH Rad3 XPD 5’ to 3’ helicase Tfb3 MAT1 CDK assembly factor Kin28 Cdk7 CDK; C-terminal domain kinase; CAK Ccl1 CycH Cyclin Rad 2 XPG Endonuclease (3’ incision); stabilizes full open complex Rad1 XPF Part of endonuclease (5’ incision) Rad10 ERCC1 Part of endonuclease (5’ incision)

33. NER and Human Genetic Diseases • Xeroderma pigmentosum • Severe light sensitivity • Severe pigmentation irregularities • Frequent neurological defects • Early onset of skin cancer at high incidence • Elevated frequency of other forms of cancer • Cockayne’s syndrome • Premature aging of some tissues • Dwarfism • Light sensitivity in some cases • Facial and limb abnormalities • Neuroligical abnormalities • Early death due to neurodegeneration • Trichothiodystrophy • Premature aging of some tissues • Sulfur deficient brittle hair • Facial abnormalities • Short stature • Ichthyosis (fish-like scales on the skin) • Light sensitivity in some cases Mitchell, Hoeijmakers and Niedernhofer (Divide and conquer: nucleotide excision repair battles cancer and ageing. Current Opinion in Cell Biology 15:232-240, 2003).

34. Recombinational Repair

35. Strand-break repair • Usually essential for cell survival • Many pathways, whose relative importance varies between and within organisms • Double-strand break repair by homologous recombination (HR) • Double-strand break repair by non-homologous end joining (NHEJ) • Single-strand break repair (SSBR)

36. Homologous Recombination is Based on the Ability of Single DNA Strands to Find Regions of Near-Perfect Homology Elsewhere in the Genome • Facilitation of Homology Searching by RecA and its Eukaryotic Homologs • Eukaryotic proteins important in this process include Rad51, Rad52, Rad54, Rad55, Rad57, Rad59. BRCA1 and BRCA2 interact with Rad51 and may regulate it.

37. Bypass synthesis corrects defect occuring in replication • Bypass polymerases with reduced fidelity can read through lesions, increasing the possiblity of inducing errors in the new DNA • Such enzymes have low processivity, only synthesizing short fragments and limiting copy errors • Excision repair can cut out damaged nucleotides and repair damage