1 DNA Repair Dr Derakhshandeh-Peykar, PhD

2 For DNA information must be transmitted intact to daughter cells

3 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

4 Accuracy is maintained by: 2-Mechanisms for correcting genetic info. in damaged DNA e.g due to chemical modifications Irradiation changes

5 1. Direct Repair - Damaged base undergoes a chemical/UV reaction Restores original structure (pro) e.g. DNA photolyase - E.coli 2-Mechanisms for correcting genetic info. in damaged DNA

6 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

7 3. Base Excision Repair (Euk/Pro) Starts at cleavage of glycosidic bond (connects base to sugar-phosphate backbone) glycosidic bond 2-Mechanisms for correcting genetic info. in damaged DNA

8 4. Nucleotide Excision Repair: (Prok: 12 bp/Euk: 28bp) - damaged DNA: excised replaced with normal DNA 2-Mechanisms for correcting genetic info. in damaged DNA

9 5. Recombinational Repair -Fills gaps in DNA : -Newly replicated DNA duplexes undergo genetic recombination Removal of damaged segment 2-Mechanisms for correcting genetic info. in damaged DNA

10 DNA REPAIR (1) Photoreactivation (aka Light Repair)

11 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

12 UV induced formation of Thymine Dimer

13 CT

14 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

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16 DNA REPAIR (2) Excision Repair (aka Dark Repair)

17 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)

18 (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

19 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

20 (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

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22 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

23 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

24 Excision Repair of Thymine dimers by UvrABC exinuclease of E.coli

25 (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)

26 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

27 DNA REPAIR (1) Photoreactivation (aka Light Repair) (2) Excision Repair (aka Dark Repair) (3) Postreplicative (Recombinational) Translesion Bypass Repair

28 DNA REPAIR (3) Postreplicative (Recombinational) Translesion Bypass Repair

29 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

30 Postreplicative (Recombinational) Repair Translesion Bypass

31 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

32 SOS Response

33 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

34 Why are DNA Repair Systems Necessary? E.coli Xeroderma Pigmentosum (XP)

35 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

36 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

37 Xeroderma Pigmentosum

38 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' side

39 Some mutations in XPB and XPD also produce signs of premature aging

40 Transcription-Coupled repair Protein: ERCC6 recognizes RNApol Mutation in gene: Cokayne Syndrom: MR Nerve disease Sensibility to sun