Section F - DNA Damage, repair and recombination

1. Section F - DNA Damage, repair and recombination

2. Contents F1 Mutagenesis Mutation, Replication fidelity, Physical mutagens, Chemical mutagens, Direct mutagenesis, Indirect mutagenesis F2 DNA damage DNA lesions, Oxidative damage, Alkylation, Bulky adducts F3 DNA repair Photoreactivation, Alkyltransferase, Excision repair, Mismatch repair, Hereditary repair defects F4 Recombination Homologous recombination, Site-specific recombination, Transposition

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

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

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

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

7. Examples of deletion mutations

8. Illustrations of five types of chromosomal mutations.

9. F1 Mutagenesis — Replication fidelity Important for preserve the genetic information from one generation to the next. • Spontaneous errors in DNA replication is very rare, one error per 1010 base in E. coli.

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

11. Proofreading by E. coli polymerase

12. 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

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

15. 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.

16. AGCTTCCTA TCGAAGGAT OH Br H • Base analog • incorporation : G O AGCTBCCTA TCGAAGGAT enol form • 1st round • ofreplication AGCTTCCTA TCGAAGGAT AGCTBCCTA TCGAGGGAT Br H : A • 2nd round • ofreplication O AGCTBCCTA TCGAAGGAT AGCTCCCTA TCGAGGGAT Keto form 5-BrU A·TG·C transition

17. 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.

18. E. colitranslession ? 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. • Some of the induced proteins, such as the UvrA and UvrB proteins, have roles in normal DNA repair pathways. • 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.

19. 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.

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

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

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

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

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

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

27. Oxidation products • 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). • The level of this damage can be INCREEASED by hydroxyl radicals from the radiolysis of H2O caused by ionizing radiation

28. 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)

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

30. 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

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

33. 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

34. F3 DNA repair — Alkyltransferase (烷基转移酶) Removes the alkyl group from mutagenic O6-alkylguanine which can base-pair with T. The alkyl group is transferred to the protein itself and inactivate it. Direct reversal of a lesion and is error-free

35. 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

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

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

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

40. 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

41. The parental strand is methylated at N6 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

42. 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.

43. 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.

44. 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

46. Diploid eukaryotes: crossing over • Homologous chromosomes line up in meiosis (when) • The nonsister chromatids exchange equivalent sections (what)

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

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

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

50. 6. Resolving Holliday junction