1. Welcome to My Molecular Biology Lecture   Li Xiaoling Office: M1623 QQ: 313320773 E-MAIL: 313320773 @qq.com

2. Content Chapter 1 Introduction Chapter 2 The Structures of DNA and RNA Chapter 10 R egulation in Eukaryotes Chapter 4 DNA Mutation and Repair Chapter 5 RNA Transcription Chapter 6 RNA Splicing Chapter 7 Translation Chapter 8 The Genetic code  Chapter 9 R egulation in prokaryotes Chapter 3 DNA Replication

3. 2015-11-25 How to learn this course well ？  To learn effectively  To preview and review  Problem-base learning  Making use of class time effectively  Active participation  Bi-directional question in class  Group discussion  Concept map  Tutorship  To call for reading, thingking and discussing of investigative learning

4. 2015-11-25 Evaluation (grading) system  Question in-class and attendance : 10 points  Group study and attendance: 20 points  Final exam: 70 points  Bonus

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

6. 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.

7. Build up a serious attitude to science!!! I absolutely do not agree with Waston et al. at the points described 3 rd & 4 th paragraphs on page 235 1. What are the more reasonable explanations for the 10 -10 mutation frequency in living organisms? 2. 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?

8. 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.

9. CHAPTER 4: DNA Mutation and Repair Molecular Biology Course 1.Replication errors and their repair 2.DNA damage 3.Repair of DNA damage

10. 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

11. 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.

12. 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

13. 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?

14. Topic 1: Replication errors and their repair CHAPTER 4 DNA Mutation and Repair 1.How the replication errors are resulted? 2.What is the nature of the replication errors? 3.How they are recognized and correctly repair?

15. 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

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

17. 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.

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

19. 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.

20. 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

21. Figure 4-2 Generation of Mutation

22. 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

23. 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

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

25. 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 (why?) (UrvD) and one of three exonucleases (why?).

26. DNA polymerase III Helicase Exonuclease,

27. 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.

28. Figure 4-5

29. Detail 2: Different exonucleases are used to remove ssDNA between the nick created by MutH and the mismatch. Figure 4-6

30. 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.

31. Topic 2: DNA dmage CHAPTER 4 DNA Mutation and Repair

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

33. Figure 4-7: Mutation due to hydrolytic damage Deamination C  U Hydrolysis creates apurinic deoxyribose Deamination 5-mC  T

34. 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? 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).

35. DNA is damaged by alkylation ( 烷基化 ), oxidation ( 氧化 ) and radiation ( 辐射 ) DNA damage Figure 4-8 G modification Alkylating chemical: Nitrosamines ( 亚硝胺 ) Reactive oxygen species (O 2 -, H 2 O 2, OH )

36. “ O 2 - ” hyperoxide “ H 2 O 2 ” Peroxide “ OH ” hydroxyl

37. Figure 4-9 Thymine dimer. UV induces a cyclobutane ( 环丁烷 ) ring between adjacent T. Radiation damage 1

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

39. 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 ).

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

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

42. Figure 4-10b Intercalating agents 溴乙非啶 二氨基吖啶 / 原黄素 吖啶, 氮蒽

43. Topic 3: Repair of DNA damage CHAPTER 4 DNA Mutation and Repair 3/22/05

44. 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

45.  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 4-1 for summary Repair of DNA damage

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

47. Photoreactivation Figure 4-11 Monomerization of thymine dimers by DNA photolyases in the presence of visible light.

48. Methyltransferase Removes the methyl group from the methylated O 6 -methylguanine. The methyl group is transferred to the protein itself, inactivating the protein. Figure 4-12

49. Base Excision repair enzyme remove damaged bases by a base- flipping mechanism Repair of DNA damage Glycosylase 1.Recognizes the damaged base 2.Removes the damaged base AP endonulease & exonulcease 3.Cleaves the abasic sugars Exonulcease/DNA polymerase/ligase 4. Works sequentially to complete the repair event.

50. Figure 4-14: base-flipping recognition by glycosylase

51. Figure 4-13: removes the damaged base and repair

52. 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 4-15:

53. 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.

54. Figure 4-16**

55. Figure 4-17. 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

56. 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 4

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

58. FIGURE 4-3 DSB repair model for homologous recombination

59. 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.

60. 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.

61. FIGURE 4-19 Crystal structure of a translesion polymerase. A Y-family polymerase found in many organisms.

62. FIGURE 4-19 Translesion DNA synthesis in E. coli

63. Summary and key points All the repair mechanisms (details) and the cause of the corresponding DNA errors and DNA damages Mismatch repair system: DNA replication errors Direct reversal of DNA damages: utraviolet (UV) irradiation induced thymine/pyrimidine dimmers---photoactivation; alkylation agents caused O 6 -methylguanine---methyl transferase. Base excision repair: the base damage by alkylation and oxidation Nucleotide excision repair: the distortion of the DNA double helix by thymine dimmer or the bulky chemical adduct ( 加合物 ) on a base. Recombination repair: double-strand breaks in DNA, errors encountered by a replication fork. Translesion synthesis allows the replication to proceed across DNA damage at a cost of error-prone replication. A different DNA polymerase is utilized. CHAPTER 4 DNA Mutation and Repair

64. Review the lecture Homework CHAPTER 4 DNA Mutation and Repair