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DNA Oxidation: Basics of Repair

Virtual Free Radical School. DNA Oxidation: Basics of Repair. Karl E. Herbert, Ph.D. & Pratibha Mistry, Ph.D. Faculty of Medicine & Biological Sciences, University of Leicester, Leicester, UK. feedback: keh3@le.ac.uk. Overview. DNA damage base excision repair ( BER )

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DNA Oxidation: Basics of Repair

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  1. Virtual Free Radical School DNA Oxidation: Basics of Repair Karl E. Herbert, Ph.D. & Pratibha Mistry, Ph.D. Faculty of Medicine & Biological Sciences, University of Leicester, Leicester, UK feedback: keh3@le.ac.uk SFRBM Education Program

  2. Overview • DNA damage • base excision repair (BER) • global genome (GG) & transcription coupled (TC) nucleotide excision repair (NER) • sanitization of the deoxynucleotide pool SFRBM Education Program

  3. Consequences of DNA damage DNA DAMAGE DNA REPAIR MECHANISMS SHORT-TERM CONSEQUENCES PHYSIOLOGICAL DYSFUNCTION CELL DEATH ABNORMAL GROWTH & METABOLISM Decreased cellular proliferation Genomic instability Defective signalling pathways Impaired protein/ gene expression LONG-TERM CONSEQUENCES Ageing Cancer Disease SFRBM Education Program

  4. Types of oxidative DNA damage • purine & pyrimidine oxidation products • abasic (AP) sites • strand breaks • see Schafer, SFRBM Education Program presentation: • http://www.medicine.uiowa.edu/frrb/ VirtualSchool/Virtual.html SFRBM Education Program

  5. BER • non-bulky lesions, abasic sites & strand breaks are repaired by BER • mono- or bifunctional lesion-specific glycosylases may initiate repair by the cleavage of the N-C1’ glycosidic bond between the base & deoxyribose- phosphate backbone leaving an abasic site • following the removal of abasic sites, short or long-patch BER resumes resulting in the repair of a single or 2-8 nucleotide gap, respectively SFRBM Education Program

  6. BER enzymes: e.g. functional diversity I MutM/fpg: formamidopyrimidine glycosylase endo VIII/Nei: endonuclease VIII OGG: 8-oxoguanine DNA glycosylase NEIL: Nei endonuclease VIII-like MPG: N-methylpurine-DNA glycosylase AlkA: 3-methyladenine DNA glycosylase II SFRBM Education Program

  7. BER enzymes: e.g. functional diversity II endo III : endonucleases III Ntg: endonuclease III-like glycosylase NTHL1: Nth endonuclease III-like 1 TDG: thymine-DNA glycosylase SFRBM Education Program

  8. ROS OH C 8-oxoG T A C Bi-functional glycosylase 8-oxoG 3’ -lyase OH C T A C A A A A A G G G G G T T T T T C C C C C G G G G G APE HO HO HO HO HO OH dGTP C C A T DNA Pol XRCC1 HO OH C T A C DNA ligase III OH C G T A C Short-patch BER SFRBM Education Program

  9. NucleicAcids 8-oxoguanine H Reactive Oxygen Species Damage hAPE: -elimination H hOGG1: -lyase hOGG1: glycosylase BER of 8-oxoG hAPE/HAP1/REF1: apurinic/apyrimidinic endonuclease SFRBM Education Program

  10. Other BER Proteins SFRBM Education Program

  11. Single-strand break HO OH C T C T A G A G C A G T C G PARP XRCC1 HO OH C T C T A G A G C A G T C G PCNA DNA Pol/ A A A G G T T C C G G A A G T C G G G T T C C G G HO HO HO HO HO OH dNTP’s C C G C G G A C T C C C A C A C A G C T T T A G T C G FEN-1 OH T C A G A G T C G C T C T A G A G C C DNA ligase I OH T C A G A G T C G Long-patch BER SFRBM Education Program

  12. DNA repair pathways & the prevention of mutations Complexities of DNA repair pathways & replication… ..specific enzymes prevent incorrect base pairing opposite DNA lesions during DNA replication Sanitization of the deoxynucleotide pool... ..specific enzymes prevent the incorporation of damaged deoxynucleotides opposite a template strand during DNA replication SFRBM Education Program

  13. 8-oxoG: 8-oxoG:A 8-oxoG:A T:A hMYH mismatch A e.g. NEIL1 8-oxoG 8-oxoG:C e.g. NEIL1 8-oxoG G:C Complexities of repair pathways & replication.. “template strand” misincorporation oxidation A/T/G   G: C good  bad X SFRBM Education Program

  14. Sanitization of the deoxynucleotide pool.. …prevents incorporation of 8-oxoG into DNA MutT: nucleoside triphosphate pyrophosphohydrolase NUDT1: nudix (nucleoside diphosphate linked moiety X)- type motif 1 hMTH1: human homolog of MutT SFRBM Education Program

  15. GG & TC NER • bulky & small lesions causing differing degrees of DNA distortion are removed by NER • NER is implicated as a back-up system for BER • different complexes initiate NER in transcribed & non-transcribed DNA • NER involves the dual incision of unwound DNA either side of the lesion by a multisubunit ATP-dependent nuclease & the release of a 24-29 oligomer • the resultant gap is filled & sealed by the action of various components including DNA polymerases & ligases SFRBM Education Program

  16. Bulky lesion Transcription-coupled Global Genomic 5’ 3’ 3’ 5’ 5’ 5’ 3’ 3’ XPC:hHR23B CSA RNA Pol II ‘DNA unwinding’: XPB, XPD 3’ 3’ 5’ 5’ CSB 24-29mer repair product XPF XPG TFIIH ‘Repair synthesis’: dNTP’s, PCNA, RFC, RPA, DNA Pol d/e, XRCC1 5’ 3’ XPA 3’ 5’ RPA 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ DNA ligase GG & TC NER SFRBM Education Program

  17. Bibliography Human DNA Repair Enzymes: http://www.cgal.icnet.uk/DNA_Repair_Genes.html Boiteux S and Radicella JP (2000). The human OGG1 gene: structure, functions and its implication in the process of carcinogenesis. Arch Biochem Biophys. 377: 1-8 Friedberg EC (1996). Relationships between DNA repair and transcription. Ann Rev Biochem. 65: 15-42 Hanawalt PC (1995). DNA repair comes of age. Mutat Res.336: 101-113 Hazra TK, Izumi T, Boldogh I, Imhoff B, Kow YW, Jaruga P, Dizdaroglu M and Mitra S (2002). Identification and characterization of a human DNA glycosylase for repair of modified bases in oxidatively damaged DNA. Proc Natl Acad SciUSA99: 3523-3528 Hoeijmakers JHJ (2001). Genome maintenance mechanisms for preventing cancer. Nature.411: 366-374 Krokan HE, Nilsen H, Skorpen F, Otterlei M and Slupphaug G (2000). Base excision repair of DNA in mammalian cells. FEBS Lett.476: 73-77 Lindahl T and Wood RD (1999). Quality control by DNA repair. Science.286: 1897-1905 Mitra S (1999). Repair of oxidative DNA damage and aging; central role of AP-endonuclease. In: Dizdaroglu and Karakaya, eds. Advances in DNA Damage and Repair, Kluwer Academic, Plenum Publishers, New York. pp 295-311 Sancar A (1996). DNA excision repair. Ann Rev Biochem.65: 43-81 SFRBM Education Program

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