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DNA Repair Dr Derakhshandeh-Peykar, PhD 1 For DNA • information must be transmitted intact to daughter cells 2 Accuracy is maintained by: 1- High fidelity in replication • 3’- exonuclease activity of DNA pol I • Uracil-DNA N-glycosylase pathway (corrects mutations from deamination of cytosine) cytosine deamination Uracil methylase Thymine 3 Accuracy is maintained by: 2-Mechanisms for correcting genetic info. in damaged DNA • e.g due to chemical modifications • Irradiation changes 4 2-Mechanisms for correcting genetic info. in damaged DNA 1. Direct Repair - Damaged base undergoes a chemical/UV reaction Restores original structure (pro) • e.g. DNA photolyase - E.coli 5 2-Mechanisms for correcting genetic info. in damaged DNA 2. Mismatch Repair (Synthesis + Repairing) • MM created by replication errors • DNA Pol III proof reading • non-homologous recombination are recognized and corrected DNA Pol III 6 2-Mechanisms for correcting genetic info. in damaged DNA 3. Base Excision Repair (Euk/Pro) • Starts at cleavage of glycosidic bond (connects base to sugar-phosphate backbone) glycosidic bond 7 2-Mechanisms for correcting genetic info. in damaged DNA 4. Nucleotide Excision Repair: (Prok: 12 bp/Euk: 28bp) - damaged DNA: • excised • replaced with normal DNA 8 2-Mechanisms for correcting genetic info. in damaged DNA 5. Recombinational Repair - Fills gaps in DNA : - Newly replicated DNA duplexes undergo genetic recombination • Removal of damaged segment 9 DNA REPAIR (1) Photoreactivation (aka Light Repair) 10 DIRECT DNA DAMAGE AND REPAIR • • • • DNA damage of a variety of sorts: A variety of irradiation (ionizing, ultraviolet, etc) U.V. induced formation of Thymine Dimmer Blocked replication and gene expression until repaired • Prohotoreactivation enzyme • Photolyase • Prokaryote 11 UV induced formation of Thymine Dimer 12 T C 13 Photoreactivation (Light Repair) • • • • • • • • PHR/PRE gene codes for photolyase with cofactor folic acid binds in dark to T dimer When light shines on cell folic acid absorbs the light (photon) uses the energy to break bond of T dimer photolyase then falls off DNA 14 15 DNA REPAIR (2) Excision Repair (aka Dark Repair) 16 Excision Repair (Dark Repair) • 3 different types of repair mechanisms • use different enzymes • (a) AP Repair (Base Excision Repair, BER) • (b) UV Damage Repair (also called NER nucleotide excision repair) • (c) Mismatch Repair (MMR) 17 (a) AP Repair (Base Excision Repair, BER) • Repair of apurinic and apyrimidinic sites on DNA • in which base: has been removed • Base removed by: – DNA glycosylases – which remove damaged bases • ung gene codes for uracil-DNA glycosylase – recognizes and removes U in DNA – by cleaving the sugar-nitrogen bond to remove the base 18 AP endonucleases: • class I nick at 3' side of AP site • class II nick at 5' side of AP site • Exonuclease removes short region of DNA • DNA Pol I and ligase fill in gap 19 (b) UV Damage Repair (also called NER - nucleotide excision repair) • It uses different enzymes • NER removes a large "patch" around the damage • Even though there may be only a single "bad" base to correct, its nucleotide is removed along with many other adjacent nucleotides • NER: UV • BER: Chemicals/Agents 20 21 NER (UV Damage Repair) • Nuclease: • can detect T dimer • nicks DNA strand on 5' end of dimer (composed of subunits coded by uvrA, uvrB and uvrC genes) • UvrA protein and ATP bind to DNA at the distortion • UvrB binds to the UvrA-DNA complex and increases specificity of UvrA-ATP complex for irradiated DNA 22 • UvrC nicks DNA 8 bases upstream and 4 or 5 bases downstream of dimer • UvrD (DNA helicase II; same as DnaB) separates strands to release 12-bp segment • DNA polymerase I now fills in gap in 5'>3' direction • ligase seals • polA - encodes DNA pol I – mutant was viable retained normal 5'>3' exo activity – only 2% of polymerase activity 23 Excision Repair of Thymine dimers by UvrABC exinuclease of E.coli 24 (c) Mismatch Repair (MMR) • Accounts for 99% of all repairs • Mismatch from replication • behind replication fork • Two ways to correct mistakes made during replication: 1) 3'>5' exonuclease - proofreading 2) Mismatch repair • mutL • mutS • mutH • and mutU (same UvrD) gene products involved (mut for mutator because if gene is mutated, cell has increased levels of spontaneous mutations) 25 How does system recognize progeny strand rather than parent strand as one with mismatch? • Because of methylation • DNA methylase (coded for by dam [DNA adenine methylase] locus) • methylates 5'-GATC-3' sequence in DNA at A residue • Mismatch from replication recognized by mutL and mutS gene products • mutH gene product nicks DNA strand (progeny strand) on either side of mismatch • DNA helicase II from mutU gene (also called uvrD gene) • unwinds DNA duplex and releases nicked region • Gap filled in by DNA Pol I and ligase 26 DNA REPAIR • (1) Photoreactivation (aka Light Repair) • (2) Excision Repair (aka Dark Repair) • (3) Postreplicative (Recombinational) Translesion Bypass Repair 27 DNA REPAIR (3) Postreplicative (Recombinational) Translesion Bypass Repair 28 Translesion bypass • Recent research in bacteria, yeast and mammalian cells • most of the mutations arise by transletion bypass • when highly processive semiconservative DNA replication is arrested at DNA lesions • translesion synthesis (TLS) polymerases allows them to insert nucleotides opposite DNA lesions, but at the expense of frequent misincorporations 29 Postreplicative (Recombinational) Repair Translesion Bypass 30 SOS response • If T dimer is not repaired • DNA Pol III can't make complementary strand during replication • leaves large gap (800 bases) • Gap may be repaired by enzymes in recombination system • RecA - coats ssDNA • it also acts as autocatalysis of LexA repressor • recA mutants - very UV-sensitive • Now have sister-strand exchange - a type of recombination: Translesion bypass • Postreplicative repair is part of SOS response 31 SOS Response 32 • LexA normally represses about 18 genes sulA and sulB, activated by SOS system • inhibit cell division in order to increase amount of time cell has to repair damage before replication • Each gene has SOS box in promoter • LexA binds SOS box to repress expression • RecA : LexA catalyses its own breakdown when RecA is stimulated by ssDNA • due to RecA binding ssDNA in lesions • could then bind to DNA Pol III complex passing through this area of the DNA • RecA no longer catalyzes cleavage of LexA (which is still being made) • so uncleaved LexA accumulates and turns the SOS system off 33 Why are DNA Repair Systems Necessary? • E.coli • Xeroderma Pigmentosum (XP) 34 E.coli • repairing thymine dimers • important to bacteria • an E. coli strain that is: – phr (no photoreactivation) – recA (no translesion by pass or SOS) – uvrA (no excision repair) is killed by a single thymine dimer 35 Xeroderma Pigmentosum (XP) • XP is a rare inherited disease of humans • predisposes the patient to: – pigmented lesions on areas of the skin exposed to the sun – an elevated incidence of skin cancer 36 Xeroderma Pigmentosum 37 • It turns out that XP can be caused by mutations in any one of several genes • all of which have roles to play in NER • Some of them: • XPA, which encodes a protein that binds the damaged site • assemble the other proteins needed for NER • XPB and XPD, which are part of TFIIH (Helicase) • XPF, which cuts the backbone on the 5' side of the damage • XPG, which cuts the backbone on the 3' 38 side Some mutations in XPB and XPD also produce signs of premature aging 39 Transcription-Coupled repair • Protein: ERCC6 recognizes RNApol Mutation in gene: Cokayne Syndrom: MR Nerve disease Sensibility to sun 40