Slow DNA rejoining in ultraviolet-irradiated human diploid fibroblasts treated with the mitogens trypsin and insulin.
Normally in mammalian cells the postincision steps of UV-induced excision repair are much more rapid than the recognition of damage and incision. This means that at any one time the level of repair-generated single-stranded DNA breaks is very low. Here we report that detectable levels of DNA breaks accumulate in quiescent human fibroblasts which are UV irradiated a few hours after replating in conditions that stimulate progress through the cell cycle. Most DNA breaks accumulate in cultures trypsinized and seeded in medium supplemented with insulin, and irradiated in early G1. Because trypsin and insulin have no effect on UV-induced incision rates, as measured by DNA break accumulation in the presence of DNA synthesis inhibitors, we argue that our ability to detect incomplete repair-sites is due to a significant reduction in the rate of gap sealing indicative of a shift in the steady state of excision repair. Provision of DNA precursors prevents the enhancing effect of trypsin and insulin on the accumulation of DNA breaks, implying that these agents affect DNA precursor metabolism. Perturbation of the repair process, which leads to the accumulation of 1500-2000 DNA breaks/genome, is also associated with other effects including increased lethality, the appearance of double-strand breaks and the loss of NAD, the last effect presumably arising as a consequence of break-stimulated poly(ADPR) transferase activity. Addition of 3-amino-benzamide, an inhibitor of poly(ADPR) synthesis, completely blocks the decline in NAD levels, but does not change the rate of sealing of the accumulated DNA breaks. These results strongly suggest that ligation is largely, if not entirely, independent of ADP ribosylation in this system.
Squires
S
S
Elliott
G C
GC
Johnson
R T
RT
D000246:Adenosine Diphosphate Ribose / Q000096:biosynthesis; D002470:Cell Survival / Q000528:radiation effects; D004247:DNA / Q000096:biosynthesis / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects*; D003854:Deoxyribonucleotides / Q000494:pharmacology; D005347:Fibroblasts; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D009243:NAD / Q000032:analysis; D012265:Ribonucleotides / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology*; D014466:Ultraviolet Rays
D003854:Deoxyribonucleotides; D007328:Insulin; D012265:Ribonucleotides; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D004247:DNA; D014357:Trypsin