Poly(ADP-ribose) polymerase-1 (PARP-1) is required in murine cell lines for base excision repair of oxidative DNA damage in the absence of DNA polymerase beta.
Oxidative DNA base damage is mainly corrected by the base excision repair (BER) pathway, which can be divided into two subpathways depending on the length of the resynthetized patch, either one nucleotide for short patch BER or several nucleotides for long patch BER. The role of proteins in the course of BER processes has been investigated in vitro using purified enzymes and cell-free extracts. In this study, we have investigated the repair of 8-oxo-7,8-dihydroguanine (8-oxoG) in vivo using wild-type, polymerase beta(-/-) (Polbeta(-/-)), poly(ADP-ribose) polymerase-1(-/-) (PARP-1(-/-)), and Polbeta(-/-)PARP-1(-/-) 3T3 cell lines. We used non replicating plasmids containing a 8-oxoG:C base pair to study the repair of the lesion located in a transcribed sequence (TS) or in a non-transcribed sequence (NTS). The results show that 8-oxoG repair in TS is not significantly impaired in cells deficient in Polbeta or PARP-1 or both. Whereas 8-oxoG repair in NTS is normal in Polbeta-null cells, it is delayed in PARP-1-null cells and greatly impaired in cells deficient in both Polbeta and PARP-1. The removal of 8-oxoG and presumably the cleavage at the resulting apurinic/apyrimidinic site are not affected in the PARP-1(-/-)Polbeta(-/-) cell lines. However, 8-oxoG repair is incomplete, yielding plasmid molecules with a nick at the site of the lesion. Therefore, PARP-1(-/-)Polbeta(-/-) cell lines cannot perform 5'-dRP removal and/or DNA repair synthesis. Furthermore, the poly(ADP-ribosyl)ation activity of PARP-1 is essential for 8-oxoG repair in a Polbeta(-/-) context, because expression of the catalytically inactive PARP-1 (E988K) mutant does not restore 8-oxoG repair, whereas an wild type PARP-1 does.
Le Page
Florence
F
Commissariat à l'Energie Atomique (CEA), Direction des Sciences du Vivant, Département de Radiobiologie et Radiopathologie, Unité Mixte de Recherche 217 CNRS-CEA Radiobiologie Moléculaire et Cellulaire, 92265 Fontenay aux Roses, France. lepage@dsvidf.fr
Schreiber
Valerie
V
Dherin
Claudine
C
De Murcia
Gilbert
G
Boiteux
Serge
S
D016475:3T3 Cells; D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002474:Cell-Free System; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000378:metabolism*; D004260:DNA Repair; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006151:Guanosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D009154:Mutation; D010100:Oxygen / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism / Q000502:physiology*; D013997:Time Factors
D000276:Adjuvants, Immunologic; D006151:Guanosine; C046215:8-hydroxyguanosine; D011065:Poly(ADP-ribose) Polymerases; D019951:DNA Polymerase beta; D010100:Oxygen