NAD(+)-dependent repair of damaged DNA by human cell extracts.
Rejoining of DNA single-strand breaks generated by treatment of plasmids with gamma-rays, neocarzinostatin, or bleomycin was catalyzed inefficiently by human cell extracts. The reaction was strongly promoted by the addition of NAD+, which was employed for rapid and transient synthesis of poly(ADP-ribose). The DNA rejoining reaction was accompanied by DNA repair replication, apparently due to replacement of damaged residues at termini. Selective depletion of poly(ADP-ribose) polymerase from cell extracts improved the repair of DNA exposed to a variety of DNA-damaging agents by removing the NAD+ dependence of the repair reaction. NAD(+)-promoted DNA repair by soluble cell extracts also occurred with alkylated DNA as substrate and was suppressed by 3-aminobenzamide. A similar stimulatory effect by NAD+ was observed for repair of ultraviolet-irradiated DNA, and this could be ascribed to the presence of pyrimidine hydrates as minor radiation-induced DNA lesions. No effect was observed on the sealing of gamma-irradiated DNA by supplementation of cell extracts with purified mammalian DNA ligase I or DNA ligase II. The results indicate that poly(ADP-ribose) polymerase interferes with base excision-repair processes because bound enzyme molecules block DNA strand interruptions. Release of bound poly-(ADP-ribose) polymerase following automodification, or physical removal of the protein from reaction mixtures, facilitates DNA repair.
Satoh
M S
MS
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, United Kingdom.
Poirier
G G
GG
Lindahl
T
T
D000228:Adenine Phosphoribosyltransferase / Q000378:metabolism; D000478:Alkylation; D001761:Bleomycin / Q000494:pharmacology; D002474:Cell-Free System; D002478:Cells, Cultured; D004247:DNA / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000502:physiology*; D009243:NAD / Q000378:metabolism*; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism; D014466:Ultraviolet Rays; D009353:Zinostatin / Q000494:pharmacology
D009243:NAD; D001761:Bleomycin; D011064:Poly Adenosine Diphosphate Ribose; D004247:DNA; D009353:Zinostatin; D000228:Adenine Phosphoribosyltransferase; D011088:DNA Ligases