1. Welcome Each of You to My Molecular Biology Class

2. Molecular Biology of the Gene, 5/E --- Watson et al. (2004) Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods 3/22/05

3. Part II: Maintenance of the Genome Dedicated to the structure of DNA and the processes that propagate, maintain and alter it from one cell generation to the next

4. Ch 6: The structures of DNA and RNA Ch 7: Chromosomes, chromatins and the nucleosome Ch 8: The replication of DNA Ch 9: The mutability and repair of DNA Ch 10: Homologous recombination at the molecular level Ch 11: Site-specific recombination and transposition of DNA 3/22/05

5. CHAPTER 9: The mutability and repair of DNA Molecular Biology Course

6. Different changes of DNA -behavior re-address ( 行为矫正） Chapter 8: Mutation ( 突变 ) is bad (death and unhealthy), which needs to be repaired Chapter 9: Recombination ( 重组 ) is good (diversity in a species- beautiful), which is promoted Chapter 10: Transposition ( 转座 ) is not bad, because it is not repaired. (benefit?)

7. The consequence of high rates of mutation Mutation in germ line ( 生殖细胞 ) would destroy the species Mutation in soma ( 体细胞 ) would destroy the individual. Maintenance of the correctness of the DNA sequence is definitely crucial for living organisms. Keeping the error rate as low as 10 -10 is so expensive.

8. Be fair ( 公正 ) and serious ( 严肃 ) to science The points that I absolutely do not agree with Waston et al. 1. Mutation is not good, it could not be responsible for biodiversity. 2. Transposition is different from mutation because (1) producing mechanism is different; (2) no mechanism to correct it; (2) existing in nature in a well- controlled manner (10 -5 ). ( 物种起源 )

9. Two important sources for mutation (unavoidable) Inaccuracy in DNA replication (10 -7 is not accurate enough) Errors ( 错误 ) Chemical damage to the genetic material (environment) Lesions ( 损害, 伤害 arose from spontaneous damage) Damage ( 损害, 伤害 caused by chemical agents and radiation

10. To repair an error or damage First, Detect the errors Second, Mend/repair the errors or lesions in a way to restore the original DNA sequence.

11. Questions to be addressed 1. How is the DNA mended rapidly enough to prevent errors from becoming set in the genetic material as mutation 2. How does the cell distinguish the parental strand from the daughter strand in repairing replication errors

12. 3. How does the cell restore the proper DNA sequence when the original sequence can no longer be read? 4. How does the cell deal with lesions that block replication?

13. Topic 1: Replication errors and their repair CHAPTER 9 The mutability and repair of DNA 3/22/05

14. The nature of mutations Point mutations: 1.Transitions (pyrimidine to pyrimidine, purine to purine) 2.Transversions (pyrimidine to purine, purine to pyrimidine) Replication errors and replication

15. Insertions Deletions Gross rearrangement of chromosome. These mutations might be caused by insertion by transposon or by aberrant action of cellular recombination processes.

16. Rate of spontaneous mutation at any given site on chromosomal ranges from 10 -6 to 10 -11 per round of DNA replication, with some sites being “hotspot”. Mutation-prone sequence in human genome are repeats of simple di-, tri- or tetranucleotide sequences, known as DNA microsatellites ( 微卫星 DNA). These sequences (1) are important in human genetics and disease, (2) hard to be copied accurately and highly polymorphic in the population.

17. Some replication errors escape proofreading The 3’-5’ exonuclease activity of replisome only improves the fidelity of DNA replication by a factor of 100-fold. The misincorporated nucleotide needs to be detected and replaced, otherwise it will cause mutation. Replication errors and replication

18. Figure 9-2 Generation of Mutation

19. Mismatch repair removes errors that escape proofreading Increase the accuracy of DNA synthesis for 2-3 orders of magnitudes. Two challenges: (1)rapidly find the mismatches/mispairs, (2) Accurately correct the mismatch Replication errors and replication Talking about the story of E. coli repair system

20. MutS scans the DNA, recognizing the mismatch from the distortion they cause in the DNA backbone MutS embraces the mismatch-containing DNA, inducing a pronounced kink in the DNA and a conformational change in MutS itself

21. Figure 9-4 Crystal structure of MutS MutS is a dimer. One monomer interacts with the mismatch specifically, and the other nonspecifically. DNA is kinked

22. MutS-mismatch-containing DNA complex recruits MutL, MutL activates MutH, an enzyme causing an incision or nick on one strand near the site of the mismatch. Nicking is followed by the specific helicase (?) (UrvD) and one of three exonucleases (?).

23. DNA polymerase III

24. Detail 1: How does the E. coli mismatch repair system know which of the two mismatched nucleotide to replace? The newly synthesized strand is not methylated by Dam methylase in a few minutes after the synthesis.

25. Figure 9-5

26. Detail 2: Different exonucleases are used to remove ssDNA between the nick created by MutH and the mismatch. Figure 9-6

27. Eukaryotic cells also repair mismatches and do so using homologs to MutS (MSH) and MutL (MLH). The underlying mechanisms are not the same and not well understood.

28. Topic 2: DNA dmage CHAPTER 9 The mutability and repair of DNA 3/22/05

29. DNA undergoes damage spontaneously ( 自发的 ) from hydrolysis and deamination Resulted from the action of water DNA damage

30. Figure 9-7: Mutation due to hydrolytic damage Deamination C  U Hydrolysis creates apurinic deoxyribose Deamination 5-mC  T

31. The presence of U and apurinic deoxyribose in DNA resulted from hydrolytic reactions is regarded as unnatural, thus is easily be recognized and repaired. Explaining why DNA contains T instead of U (?) Can 5-mC  T lesion be repaired?

32. DNA is damaged by alkylation ( 烷基化 ), oxidation ( 氧化 ) and radiation ( 辐射 ) DNA damage Figure 9-8 G modification Nitrosamines ( 亚硝胺 ) Reactive oxygen species (O 2 -, H 2 O 2, OH )

33. Figure 9-9 Thymine dimer. UV induces a cyclobutane ( 环丁烷 ) ring between adjacent T.

34. Gamma radiation and X-rays (ionizing radiation) cause double- strand breaks and are particularly hazardous (hard to be repaired).

35. Mutations are also caused by base analogs and intercalating agents DNA damage  Base analogs: similar enough to the normal bases to be processed by cells and incorporated into DNA during replication.  But they base pair differently, leading to mistake during replication.  The most mutagenic base anolog is 5-bromouracil.

37.  Intercalating agents are flat molecules containing several polycyclic rings that interact with the normal bases in DNA through hydrogen bonds and base stacking.

39. Topic 3: Repair of DNA damage CHAPTER 9 The mutability and repair of DNA 3/22/05

40. Two consequence of DNA damage Some damages, such as thymine dimer, nick or breaks in the DNA backbone, create impediments to replication or transcription Some damages creates altered bases that has no effect on replication but cause mispairing, which in turn can be converted to mutation. Repair of DNA damage

41.  Direct reversal of DNA damage by photoreactivation ( 光活化作用 ) and alkyltransferase ( 烷基转移酶 )  Base excision repair ( 切割修复 )  Nucleotide excision repair  Recombination (DSB) repairs  Translesion DNA synthesis Mechanisms to repair a damage See Table 9-1 for summary Repair of DNA damage

42. Direct reversal of DNA damage Error-free repair Repair of DNA damage

43. Photoreactivation Figure 9-11 Monomerization of thymine dimers by DNA photolyases in the presence of visible light.

44. Methyltransferase Removes the methyl group from the methylated O 6 -methylguanine. The methyl group is transferred to the protein itself, inactivating the protein. Figure 9-12

45. Base Excision repair enzyme remove damaged bases by a base- flipping mechanism Repair of DNA damage Glycosylase Recognizes the damaged base Removes the damaged base AP endonulease & exonulcease 3.Cleaves the abasic sugars Exonulcease/DNA polymerase/ligase 4. Works sequentially to complete the repair event.

46. Figure 9-14: base-flipping recognition

47. Figure 9-13: removes the damaged base and repair

48. oxoG:A repair. A glycosylase recognizes the mispair and removes A. A fail-safe glycosylase also removes T from T:G mispairs, as if it knows how T is produced? Fail-safe systems ( 最后保险系统 ) Figure 9-15:

49. Nucleotide Excision repair enzymes cleave damaged DNA on either side of the lesion Repair of DNA damage 1.Recognize distortions to the shape of the DNA double helix 2.Remove a short single- stranded segment that includes the lesion. 3.DNA polymerase/ligase fill in the gap.

50. Figure 9-16**

51. Figure 9-17. Transcription- couple repair: nucleotide excision repair (NER) system is capable of rescuing RNA polymerase that has been arrested by the presence of lesions in the DNA template TFIIH

52. Recombination repairs DNA breaks by retrieving sequence information from undamaged DNA Repair of DNA damage Double-strand break (DSB) repair pathway Details are in chapter 10

53. Figure 10-4. Damage in the DNA template can lead to DSB formation during replication

54. FIGURE 10-3 DSB repair model for homologous recombination

55. Translesion DNA synthesis enables replication to proceed across DNA damage Repair of DNA damage  Error-prone repair***  Occurs when the above repairs are not efficient enough so that a replicating polymerase encounters a lesion  Translesion synthesis is also called a fail-safe or last resort mechanism.

56. 1.Translesion synthesis is catalyzed by a specialized class of DNA polymerases that synthesize DNA directly across the damage site. 2.Translesion polymerase is produced by cell in response to the DNA damage 3.Translesion polymerases are expressed as part of the SOS response pathway.

57. FIGURE 9-19 Crystal structure of a translesion polymerase. A Y-family polymerase found in many organisms.

58. FIGURE 9-19 Translesion DNA synthesis in E. coli

59. 重点 3/25/05 掌握 slides 里的内容。 CHAPTER 9 The mutability and repair of DNA

60. Review the lecture Homework CHAPTER 9 The mutability and repair of DNA