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Molecular Biology Course

Molecular Biology Course. SECTION F DNA damage, repair, and recombination. link. DNA damage, repair & recombination. F1 Mutagenesis ( 诱变）. Mutation: replication fidelity, mutagens, mutagenesis. F2 DNA damage. DNA lesions: oxidative damage, alkylation, bulky adducts.

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Molecular Biology Course

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  1. Molecular Biology Course SECTION F DNA damage, repair, and recombination link

  2. DNA damage, repair & recombination F1 Mutagenesis (诱变） Mutation: replication fidelity, mutagens, mutagenesis F2 DNA damage DNA lesions: oxidative damage, alkylation, bulky adducts F3 DNA repair Photoreaction, alkyltransferase, excision repair, mismatch repair, hereditary repair defects F4 Recombination Homologous recombination, site-specific recombination, transposition

  3. DNA damage, repair & recombination F1 Mutagenesis (诱变） Mutation Replication fidelity Mutagens: chemical & physical Mutagenesis: direct & indirect

  4. Mutation （突变） Replication Fidelity (复制的精确性） Mutagenesis Mutagens （诱变剂） back

  5. F1 Mutaagenesis F1-1 Mutation Permanent, heritablealterations 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

  6. F1 Mutaagenesis Point mutation (a single base change) Transition: Purine or pyrimidine is replaced by the other AG T C 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

  7. F1 Mutaagenesis 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

  8. F1 Mutaagenesis 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.

  9. Examples of deletion mutations

  10. F1 Mutaagenesis F1-2 Replication fidelity Important for preserve the genetic information from one generation to the next • Mutation relevant • Spontaneous errors in DNA replication is very rare, one error per 1010 base in E. coli.

  11. F1 Mutaagenesis 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)

  12. F1 Mutaagenesis Proofreading by E. coli polymerase

  13. F1 Mutaagenesis F1-3&4: Mutagens Mutation relevant Cause DNA damage that can be converted to mutations.

  14. F1 Mutaagenesis Physical mutagens High-energy ionizing radiation: X-rays and g-rays  strand breaks and base/sugar destruction Nonionizing radiation : UV light pyrimidine dimers Chemical mutagens Base analogs: direct mutagenesis Nitrous acid: deaminates C to produce U Alkylating agents Arylating agents Lesions-indirect mutagenesis (F2)

  15. Base analogs:derivatives of the normal basesincorporated in DNA, altering base pairing properties. Nitrous acid: deaminates Cto produce U, resulting in G·C  A·U

  16. F1 Mutaagenesis F1-3&4: Mutagenesis The molecular process in which the mutation is generated. Note: the great majority of lesions introduced by chemical and physical mutagens are repaired by one or more of the error-free DNA repair mechanisms before the lesions is encounter by a replication fork

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

  18. F1 Mutaagenesis AGCTTCCTA TCGAAGGAT Br Br • Base analog • incorporation AGCTBCCTA TCGAAGGAT • 1st round • of replication AGCTTCCTA TCGAAGGAT AGCTBCCTA TCGAGGGAT H • 2nd round • of replication O Keto form AGCTBCCTA TCGAAGGAT AGCTCCCTA TCGAGGGAT OH H : G O enol form : A 5-BrU A·TG·C transition

  19. F1 Mutaagenesis Indirect mutagenesis The mutation is introduced as a result of an error-prone repair. Translesion 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.

  20. F1 Mutaagenesis • 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. • 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. back

  21. Proper base pairing is often impossible and not strictly required at the site of a lesion because of the SOS response proteins, thistranslesion 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. F DNA damage, repair and recombination 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) Error-free Repairing Direct mutagenesis Indirect mutagenesis mutations Completely repaired

  23. DNA damage, repair & recombination F2DNA damage DNA lesions: oxidative damage Alkylation bulky adducts

  24. DNA damage, repair & recombination 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 Normal condition • Increased by • ionizing radiation • (physical mutagens) Alkylation (烷基化作用） Alkylating agents (Chemical mutagens)

  25. DNA damage, repair & recombination F2-1 DNA lessions An alteration to the normal chemical or physical structure of the DNA

  26. DNA lessions 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

  27. Spontaneous DNA lesions • Inherent chemical reactivity of the DNA • The presence of normal, reactive chemical species within the cell • Deamination (转氨作用）: • CU • methylcytosine T, hard to be detected • Depurination (脱瞟呤作用) : break of the glycosylic bond, non-coding lesion. • Depyrimidine (脱嘧啶作用) back

  28. DNA lessions Chemical reactivity of bases is responsible for some DNA lesion

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

  30. DNA damage, repair & recombination F2-2 Oxidative damage DNA lesions caused by reactive oxygen species such as superoxide and hydroxyl radicals

  31. Oxidation products • DNA damage • 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

  32. DNA damage, repair & recombination F2-3 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)

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

  34. DNA damage, repair & recombination F2-4 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

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

  36. DNA damage, repair & recombination F3DNA repair Photoreactivation (光活化作用) Alkyltransferase (烷基转移酶) Exision repair (切割修复) Mismatch repair (错配修复) Hereditary repair defects (遗传修复缺陷)

  37. DNA damage, repair & recombination F3-1: Photoreactivation Monomerization of cyclobutane pyrimidine dimers by DNA photolyases in the presence of visible light Direct reversal of a lesion and is error-free

  38. DNA damage, repair & recombination F3-2: Alkyltransferase 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

  39. DNA repair 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

  40. DNA damage, repair & recombination F3-3: Excision repair • Includs nucleotide excision repair (NER) and base excision repair (BER). • Is a ubiquitous mechanism repairing a variety of lesions. • Error-free repair

  41. DNA repair 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

  42. DNA repair 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

  43. DNA damage, repair & recombination F3-3: Mismatch repair A specialized form of excision repair which deals with any base mispairs produced during replication and which have escaped proofreading error-free

  44. 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 back

  45. DNA damage, repair & recombination F4Recombination Homologous recombination Site-specific recombination Transposition Mutation Relevance An important reason for variable DNA sequences among different populations of the same species

  46. DNA damage, repair & recombination F4-1 Homologous recombination ( 同源重组） 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. F4 Recombination Diploid eukaryotes: crossing over • Homologous chromosomes line up in meiosis (when) • The nonsister chromatids exchange equivalent sections (what)

  48. F4 Recombination 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)

  49. F4 Recombination 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 byRecA to form RecA-ssDNA dilaments.

  50. F4 Recombination 3. RecA-ssDNA filaments search the opposite DNA duplex for corresponding sequence (invasion). 4. form a four-branched Holliday structure 5. Branch migration back

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