2. Section F - DNA Damage, repair and recombination

3. F1 Mutagenesis Mutation, Replication fidelity, Physical mutagens, Chemical mutagens, Direct mutagenesis, Indirect mutagenesisMutationReplication fidelityPhysical mutagens Chemical mutagensDirect mutagenesisIndirect mutagenesis F2 DNA damage DNA lesions, Oxidative damage, Alkylation, Bulky adductsDNA lesionsOxidative damageAlkylationBulky adducts F3 DNA repair Photoreactivation, Alkyltransferase, Excision repair, Mismatch repair, Hereditary repair defectsPhotoreactivationAlkyltransferaseExcision repair Mismatch repairHereditary repair defects F4 Recombination Homologous recombination, Site-specific recombination, TranspositionHomologous recombinationSite-specific recombination TranspositionContents

4. F1 Mutagenesis — Mutation Mutation Mutation ： Permanent, heritable( 可遗传的 ) alterations in the base sequence of DNA. Reasons 1.Spontaneous errors in DNA replication or meiotic recombination. 2.A consequence of the damaging effects of physical or chemical mutagens on DNA.

5. TransitionTransition : Purine or pyrimidine is replaced by the other. A  GT  C Transversion : a purine is replaced by a pyrimidine or vice verse.Transversion A  T or C T  A or G G  T or C C  A or G Point mutation (a single base change) ( 颠换 ) ( 转换 )

6. Noncoding DNA Nonregulatory DNA 3 rd position of a codon Silent mutation Coding DNA  altered AA Missense mutation Phenotypic effects No Coding DNA  stop codon  truncated protein Nonsense mutation Effects of a point mutation Yes or No Yes

7. Insertions or deletions Frameshift mutations The ORF of a protein encoded gene is changed so that the C-terminal side of the mutation is completely changed. The addition or loss of one or more bases in a DNA region

8. Examples of deletion mutations

9. Illustrations of five types of chromosomal mutations.

10. F1 Mutagenesis — Replication fidelity Spontaneous errors in DNA replication is very rare, one error per 10 10 base in E. coli. Important for preserve the genetic information from one generation to the next.

11. Molecular mechanisms for the replication fidelity 1.DNA polymerase: Watson-Crick base pairing 2.3’  5’ proofreading exonuclease. 3.RNA priming: proofreading the 5’ end of the lagging strand 4.Mismatch repair (F3)

12. by E. coli polymerase Proofreading

13. F1 Mutagenesis — Physical mutagens High-energy ionizing radiation: X-rays and γ- rays  strand breaks and base/sugar destruction Nonionizing radiation : UV light  pyrimidine dimers

14. F1 Mutagenesis — Chemical mutagens Chemical mutagens: Base analogs: direct mutagenesis Nitrous acid: deaminates C to produce U Alkylating agents Arylating agents

16. F1 Mutagenesis — Direct mutagenesis Direct mutagenesis The stable, unrepaired base with altered base pairing properties in the DNA is fixed to a mutation during DNA replication.

17. 5-BrU : G : A enol form Br OH H O Br Keto form H O AGCTTCCTA TCGAAGGAT AGCTBCCTA TCGAAGGAT 1.Base analog incorporation AGCTBCCTA TCGAGGGAT AGCTTCCTA TCGAAGGAT 2.1st round of replication AGCTBCCTA TCGAAGGAT AGCTCCCTA TCGAGGGAT 3.2nd round of replication A·T  G·C transition

18. F1 Mutagenesis — Indirect mutagenesis Indirect mutagenesis The mutation is introduced as a result of an error-prone repair. Translation DNA synthesis to maintain the DNA integrity but not the sequence accuracy: when damage occurs immediately ahead of an advancing fork, which is unsuitable for recombination repair (F4), the daughter strand is synthesized regardless of the the base identity of the damaged sites of the parental DNA.

19. E. coli translession ? replication: SOS response: Higher levels of DNA damage effectively inhibit DNA replication and trigger a stress response in the cell, involving a regulated increase (induction) in the levels of a number of proteins. This is called the SOS response. 1.Some of the induced proteins, such as the UvrA and UvrB proteins, have roles in normal DNA repair pathways. 2.A number of the induced proteins, however, are part of a specialized replication system that can REPLICATE PAST the DNA lesions that block DNA polymerase III.

20. Proper base pairing is often impossible and not strictly required at the site of a lesion because of the SOS response proteins, this translesion replication is error-prone. The resulting increase in mutagenesis does not contradict the general principle that replication accuracy is important (the resulting mutations actually kill many cells). This is the biological price that is paid, however, to overcome the general barrier to replication and permit at least a few mutant cells to survive.

22. F2 DNA damage — DNA lesions DNA lesions (DNA 损害） Oxidative damage ( 氧化损伤） Alkylation ( 烷基化作用） Bulky adducts ( 加合物） 1.Occurs under the normal conditionsOccurs under the normal conditions 2.Increased byIncreased by ionizing radiation (physical mutagens) Alkylating agents (Chemical mutagens) UV light (physical mutagens) Carcinogen (Chemical mutagens)

23. The biological effect of the unrepaired DNA lesions Lethal (cell death) Physical distortion of the local DNA structure Blocks replication and/or transcription Mutagenic Allowed to remained in the DNA A mutation could become fixed by direct or indirect mutagenesis Living cell Altered chemistry of the bases

24. DNA damage and repair Mutagen ( 诱变剂） Completely repaired DNA damage (lesions) chemical reactivity of the bases Error-free Repairing mutations Indirect mutagenesis minor or moderate Extensive, right before Replication Fork (not repairable) Direct mutagenesis

25. Chemical reactivity of bases is responsible for some DNA lesion

26. Uracil DNA glycosylase deamination --ATGCTACG-- --TACGATGC-- --ATGUTACG-- --TACGATGC-- --ATG TACG-- --TACGATGC-- U --ATGCTACG-- --TACGATGC-- Cytosine deamination and repair

27. F2 DNA damage — Oxidative damage DNA lesions caused by reactive oxygen species such as superoxide and hydroxyl radicals

28. Oxidation products 1. occurs under NORMAL conditions in all aerobic cells due to the presence of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, and the hydroxyl radicals (-OH). 2.The level of this damage can be INCREEASED by hydroxyl radicals from the radiolysis of H 2 O caused by ionizing radiation

29. F2 DNA damage — Alkylation Nucleotide modification caused by electrophilic alkylating agents such as methylmethane sulfonate （甲 基甲烷磺酸盐） and ethylnitrosourea ( 乙基亚硝基脲 )

30. 1.Electrophilic chemicals adds alkyl groups to various positions on nucleic acids 2.Distinct from those methylated by normal methylating enzymes. Alkylated bases alkylating agents

31. F2 DNA damage — Bulky adducts DNA lesions that distort the double helix and cause localized denaturation, for example pyrimidine dimers and arylating agents adducts These lesions disrupt the normal function of the DNA

33. Cyclobutane pyrimidine dimer( 嘧啶二聚体 ) Guanine adduct of benzo[a]pyrene Aromatic arylating agents Covalent adducts

34. F3 DNA repair — Photoreactivation Monomerization of cyclobutane pyrimidine dimers by DNA photolyases in the presence of visible light Direct reversal of a lesion and is error-free

35. F3 DNA repair — Alkyltransferase ( F3 DNA repair — Alkyltransferase ( 烷基 转移酶 ) Direct reversal of a lesion and is error-free Removes the alkyl group from mutagenic O 6 -alkylguanine which can base-pair with T. The alkyl group is transferred to the protein itself and inactivate it.

36. The response is adaptive because it is induced in E. coli by low levels of alkylating agents and gives increased protection against the lethal and mutagenic effects of the high doses

37. F3 DNA repair — Excision repair 1.Includs nucleotide excision repair (NER) and base excision repair (BER). 2.Is a ubiquitous mechanism repairing a variety of lesions. 3.Error-free repair

39. Nucleotide excision repair 1.An endonuclease cleaves DNA a precise number of bases on both sides of the lesions (UvrABC endonulcease removes pyrimidine dimers) 2.Excised lesion-DNA fragment is removed 3.The gap is filled by DNA polymerase I and sealed by ligase

40. Base excision repair DNA glycolases cleaves apurinic or pyrimidine site DNA polymerase DNA ligase cleaves N-glycosylic bond AP endonuclease 3’  5’ cleavage and & 5’  3’ synthesis

41. F3 DNA repair — Mismatch repair A specialized form of excision repair which deals with any base mispairs produced during replication and which have escaped proofreading error-free

42. The parental strand is methylated at N 6 position of all As in GATC sites, but methylation of the daughter strand lag a few minutes after replication MutH/MutS recognize the mismatched base pair and the nearby GATC DNA helicase II, SSB, exonuclease I remove the DNA fragment including the mismatch DNA polymerase III & DNA ligase fill in the gap Expensive to keep the accuracy

43. F3 DNA repair — Hereditary repair defects Xeroderma pigmentosa, or XP, is an autosomal recessive genetic disorder of DNA repair in which the ability to repair damage caused by ultraviolet (UV) light is deficient. Xeroderma pigmentosum has an autosomal recessive pattern of inheritance.

44. The most common defect in xeroderma pigmentosum is an autosomal recessive genetic defect whereby nucleotide excision repair (NER) enzymes are mutated, leading to a reduction in or elimination of NER. Normally, damage to DNA in epidermal cells occurs during exposure to UV light. The absorption of the high energy light leads to the formation of pyrimidine dimers, namely CPD's (cyclobutane-pyrimidine-dimers) and 6- 4PP's (pyrimidine-6-4-pyrimidone photoproducts). The normal repair process entails nucleotide excision. The damage is excised by endonucleases, then the gap is filled by a DNA polymerase and "sealed" by a ligase.

45. F4 Recombination — Homologous recombination The exchange of homologous regions between two DNA moleculs Diploid eukaryotes: crossing over Haploid prokaryotes: recA-dependent, Holliday model DNA repair in replication fork

47. Diploid eukaryotesDiploid eukaryotes: crossing over 1.Homologous chromosomes line up in meiosis (when) 2.The nonsister chromatids exchange equivalent sections (what)

48. Haploid prokaryotesHaploid prokaryotes recombination Between the two homologous DNA duplex (where) 1. between the replicated portions of a partially duplicated DNA 2.between the chromosomal DNA and acquired “foreign” DNA Holliday model (How)

49. 2.Nicks made near Chi (GCTGGTGG) sites by a nuclease. 3. ssDNA carrying the 5’ ends of the nicks is coated by RecA to form RecA-ssDNA dilaments. recA-dependent bacterial homologous recombination 1.Homologous DNA pairs 3’5’ 3’5’ 3’ 5’

50. 3.RecA-ssDNA filaments search the opposite DNA duplex for corresponding sequence (invasion). 4.form a four- branched Holliday structure 5.Branch migration

51. 6.Resolving Holliday junction

53. RuvAB is an asymmetric complex that promotes branch migration of a Holliday junction.

54. Recombination based DNA repair at replication fork a.Replication encounters a DNA lesion b.Skip the lesion & re- initiate on the side of the lesion c.Fill the daughter strand gap by replacing it with the corresponding section from the parental sister strand d.post-replication repair of the left lesion

55. F4 Recombination — Site-specific recombination 1.Exchange of non-homologous but specific pieces of DNA (what) 2.Mediated by proteins that recognize specific DNA sequences. (how)

56. 1.l-encoded integrase (Int): makes staggered cuts in the specific sites 2.Int and IHF (integration host factor encoded by bacteria): recombination and insertion 3.l-encoded excisionase (XIS): excision of the phage DNA Site-specific recombination: bacteriophage  insertion Site-specific recombination: bacteriophage  insertion

57. Site-specific recombination: Antibody diversity Site-specific recombination: Antibody diversity H and L are all encoded by three gene segments: V, D, J VDJ Two heavy chains (H) 250155 Two light chains (L) 2504 Enormous number (>10 8 ) of different H and L gene sequences can be produced by such a recombination

58. F4 Recombination — Transposition 1.Requires no homology between sequences nor site-specific 2.Relatively inefficient 3.Require transposase encoded by the transposon （转座 子）

60. Various transposons: In E. coli: IS elements/insertion sequence, 1-2 kb, comprise a transposase gene flanked by a short inverted terminal repeats Tn transposon series carry transposition elements and b-lactamase (penicillin resistance) Eukaryotic transposons, many are retrotransposons: Yeast Ty element encodes a protein similar to RT (reverse transcriptase)

61. Simplified Transposition process

62. Multiple choice questions 1. Per nucleotide incorporated, the spontaneous mutation frequency in E. coli is. A 1 in 106. B 1 in 108. C 1 in 109. D 1 in 1010. 2. The action of hydroxyl radicals on DNA generates a significant amount of. A pyrimidine dimmers. B 8-oxoguanine. C O6- methylguanine. D 7-hydroxymethylguanine.

63. 3. In methyl-directed mismatch repair in E. coli, the daughter strand containing the mismatched base is nicked by. A MutH endonuclease. B UvrABC endonuclease. C AP endonuclease. D 3' to 5' exonuclease. 4. Illegitimate recombination is another name for. A site-specific recombination. B transposition. C homologous recombination. D translesion DNA synthesis.

64. 5. The excision repair of UV-induced DNA damage is defective in individuals suffering from. A hereditary nonpolyposis colon cancer. B Crohn's disease. C classical xeroderma pigmentosum. D xeroderma pigmentosum variant.

65. THANK YOU !