Ch 6: The structures of DNA and RNA Ch 7: Chromosomes, chromatins and the nucleosome

1. 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

2. Teaching arrangement: Lecture 1: (1) Introduction to DNA alterations and DNA mutations (2) Replication errors and mismatch repair. Lecture 2: (1) Review replication errors and mismatch repair;

3. 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?)

4. 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.

5. Build up a serious attitude to science!!! I absolutely do not agree with Waston et al. at the points described 3rd & 4th paragraphs on page 235 • What are the more reasonable explanations for the 10-10 mutation frequency in living organisms? • What are the evidences that such a low mutation rate can drive the evolvement of a new species if the cell changes are known harmful?

6. Listen to the nature • Mutation is not good and is naturally repaired, thus it could not be responsible for biodiversity. • Recombination is good and naturally promoted; it is responsible for diversity inside of species. • Transposition is different from mutation and recombination because (1) producing mechanism is different; (2) no mechanism to correct it; (3) existing in nature in a well-controlled manner (10-5). Not repaired but controlled.

7. CHAPTER 9: The mutability and repair of DNA • Molecular Biology Course • Replication errors and their repair • DNA damage • Repair of DNA damage

8. 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

9. 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.

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

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

12. CHAPTER 9 The mutability and repair of DNA Topic 1: Replication errors and their repair • How the replication errors are resulted? • What is the nature of the replication errors? • How they are recognized and correctly repair?

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

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

15. 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.

16. Each bases has its preferred tautomeric form (Related to Ch 9) Ch 6 DNA STRUCTURE (2)

17. The strictness of the rules for “Waston-Crick” pairing derives from the complementarity both of shape and of hydrogen bonding properties between adenine and thymine and between guanine and cytosine.

18. Some replication errors escape proofreading Replication errors and replication 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.

19. Figure 9-2 Generation of Mutation

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

21. 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

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

23. MutS-mismatch-containing DNA complex recruitsMutL,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 (why?) (UrvD) and one of three exonucleases (why?).

24. Helicase Exonuclease, DNA polymerase III

25. 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.

26. Figure 9-5

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

28. 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.

29. CHAPTER 9 The mutability and repair of DNA Topic 2: DNA dmage

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

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

32. Vertebrate DNA frequently contains 5-methyl cytosine in place of cytosine as a result of the action of methyl transferase. This modified base plays a role in the transcriptional silencing (Ch 17). The presence of U and apurinic deoxyribose in DNA resulted from hydrolytic reactions is regarded as unnatural, thus is easily be recognized and repaired. Can 5-mC  T lesion be repaired?

33. DNA is damaged by alkylation (烷基化), oxidation (氧化) and radiation (辐射) DNA damage Alkylating chemical: Nitrosamines (亚硝胺) Reactive oxygen species (O2-, H2O2, OH•) Figure 9-8 G modification

34. “O2-” hyperoxide “H2O2” Peroxide “OH•” hydroxyl

35. Figure 9-9 Thymine dimer. UV induces a cyclobutane (环丁烷) ring between adjacent T. Radiation damage 1

36. Gamma radiation and X-rays (ionizing radiation) cause double-strand breaks and are particularly hazardous (hard to be repaired). Radiation damage 2

37. Mutations are also caused by base analogs (碱基类似物) and intercalating agents (嵌入剂) 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 mispairing during replication. • The most mutagenic base analog is 5-bromoUracil (5-BrU) (溴尿嘧啶).

38. 酮异构体 烯醇异构体 Figure 9-10a Base analogues Figure 3-33 G-U pair

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

40. 溴乙非啶 吖啶, 氮蒽 二氨基吖啶/原黄素 Figure 9-10b Intercalating agents

41. CHAPTER 9 The mutability and repair of DNA Topic 3: Repair of DNA damage

42. Two consequence of DNA damage Repair 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.

43. See Table 9-1 for summary Mechanisms to repair a damage Repair of DNA damage • 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

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

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

46. Figure 9-12 Methyltransferase Removes the methyl group from the methylated O6-methylguanine. The methyl group is transferred to the protein itself, inactivating the protein.

47. 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.

48. Figure 9-14: base-flipping recognition by glycosylase

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

50. Fail-safe systems (最后保险系统) Figure 9-15: 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.