Virtual Free Radical School DNA Oxidation Basics of
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, UK feedback: keh 3@le. ac. uk DNA Oxidation: Repair 5/2003 SFRBM Education Program 1
Overview • DNA damage • base excision repair (BER) • global genome (GG) & transcription coupled (TC) nucleotide excision repair (NER) • sanitization of the deoxynucleotide pool DNA Oxidation: Repair 5/2003 SFRBM Education Program 2
Consequences of DNA damage DNA DAMAGE DNA REPAIR MECHANISMS SHORT-TERM CONSEQUENCES PHYSIOLOGICAL DYSFUNCTION Decreased cellular proliferation CELL DEATH Genomic instability ABNORMAL GROWTH & METABOLISM Defective signalling pathways Impaired protein/ gene expression LONG-TERM CONSEQUENCES Ageing DNA Oxidation: Repair 5/2003 Cancer SFRBM Education Program Disease 3
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/ Virtual. School/Virtual. html DNA Oxidation: Repair 5/2003 SFRBM Education Program 4
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-C 1’ glycosidic bond between the base & deoxyribosephosphate 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 DNA Oxidation: Repair 5/2003 SFRBM Education Program 5
BER enzymes: e. g. functional diversity I Oxidized & ring opened purines: e. g. 8 -oxo. G, fapy. G E. coli S. cerevisiae H. sapiens Mut. M (fpg) y. OGG 1&2 h. OGG 1 endo VIII (Nei) _ _ h. NEIL 1 Alk. A (not oxo. G) _ _ h. MPG Mut. M/fpg: formamidopyrimidine glycosylase endo VIII/Nei: endonuclease VIII OGG: 8 -oxoguanine DNA glycosylase NEIL: Nei endonuclease VIII-like MPG: N-methylpurine-DNA glycosylase Alk. A: 3 -methyladenine DNA glycosylase II DNA Oxidation: Repair 5/2003 SFRBM Education Program 6
BER enzymes: e. g. functional diversity II Oxidized & ring opened pyrimidines: e. g. T-glycol, fapy E. coli S. cerevisiae H. sapiens endo III (Nth) Ntg 1&2 h. NTHL 1 endo VIII (Nei) _ _ _ _ _ Alk. A h. NEIL 1 -3 _ _ _ _ _ h. TDG _ _ _ endo III : endonucleases III Ntg: endonuclease III-like glycosylase NTHL 1: Nth endonuclease III-like 1 TDG: thymine-DNA glycosylase DNA Oxidation: Repair 5/2003 SFRBM Education Program 7
Short-patch BER ROS OH C G 8 -oxo. G C A T C G T A HO Bi-functional glycosylase 8 -oxo. G 3’ -lyase OH C G A T C G C T A HO APE OH C G A T C C G T A HO DNA Pol XRCC 1 d. GTP HO OH C G A T C C G T A HO DNA ligase III OH C G A T G C C G T A HO DNA Oxidation: Repair 5/2003 SFRBM Education Program 8
BER of 8 -oxo. G Reactive Oxygen Species Damage Nucleic Acids 8 -oxoguanine H h. APE: -elimination H h. OGG 1: -lyase h. OGG 1: glycosylase h. APE/HAP 1/REF 1: apurinic/apyrimidinic endonuclease DNA Oxidation: Repair 5/2003 SFRBM Education Program 9
Other BER Proteins Removal of abasic sites APE 1 AP endonuclease (HAP 1, REF 1) Gap filling PCNA Proliferating cell nuclear antigen DNA polymerase Sealing the nick XRCC 1 LIG 3 DNA Oxidation: Repair 5/2003 X-ray cross-complementing protein & ligase accessory factor DNA ligase SFRBM Education Program 10
Long-patch BER Single-strand break H O C G A T G C G T A C G A T G C C G T A O H H O PARP C G A T H O XRCC 1 G C G T A C G A T G C O H H O T C PCNA A DNA Pol / G C d. NTP’s C G A T G C C G A G T C O H FEN-1 H O T C A G C C G A T G C C G T A O H H O T C A G DNA ligase I C H O DNA Oxidation: Repair 5/2003 C G A T G C C G T A C G A T G C SFRBM Education Program C G T A O H 11
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 DNA Oxidation: Repair 5/2003 SFRBM Education Program 12
Complexities of repair pathways & replication. . “template strand” oxidation G: 8 -oxo. G: A misincorporation A/T/G C 8 -oxo. G: A h. MYH A mismatch 8 -oxo. G e. g. NEIL 1 T: A 8 -oxo. G: C e. g. NEIL 1 8 -oxo. G G: C good DNA Oxidation: Repair 5/2003 bad X SFRBM Education Program 13
Sanitization of the deoxynucleotide pool. . …prevents incorporation of 8 -oxo. G into DNA E. coli H. sapiens substrate Mut. T NUDT 1 (h. MTH 1) 8 -oxod. GTP Mut. T: nucleoside triphosphate pyrophosphohydrolase NUDT 1: nudix (nucleoside diphosphate linked moiety X)type motif 1 h. MTH 1: human homolog of Mut. T DNA Oxidation: Repair 5/2003 SFRBM Education Program 14
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 DNA Oxidation: Repair 5/2003 SFRBM Education Program 15
GG & TC NER Bulky lesion 5’ 3’ 3’ 5’ Transcription-coupled Global Genomic XPC: h. HR 23 B RNA Pol II CSA CSB 5’ 3’ 3’ 5’ ‘DNA unwinding’: XPB, XPD XPF XPG TFIIH 5’ 3’ XPA 3’ 5’ RPA 24 -29 mer repair product ‘Repair synthesis’: d. NTP’s, PCNA, RFC, RPA, DNA Pol / , XRCC 1 5’ 3’ 3’ 5’ DNA ligase 5’ 3’ 3’ 5’ DNA Oxidation: Repair 5/2003 SFRBM Education Program 16
Bibliography Human DNA Repair Enzymes: http: //www. cgal. icnet. uk/DNA_Repair_Genes. html Boiteux S and Radicella JP (2000). The human OGG 1 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 DNA Oxidation: Repair 5/2003 SFRBM Education Program 17
- Slides: 17