Repair rates of R-band, G-band and C-band DNA in murine and human cultured cells.
Repair of cyclobutane pyrimidine dimers (CPDs) in cultured neonatal human fibroblasts and in Mus spretus x M. castaneus F1 neonatal skin fibroblasts was analyzed after UVC-irradiation by cleavage with T4 endonuclease V cyclopyrimidine dimer glycosylase, alkaline-agarose gel electrophoresis, and Southern blotting. The blots were sequentially probed with 32P-labeled Alu, or B2, to preferentially illuminate R-band DNA, by L1 to preferentially illuminate G-band DNA, and by satellite DNA to illuminate C-band DNA. These three different DNA populations showed slightly different global nucleotide excision repair rates that are in the order of speed, R-band DNA > G-band DNA > C-band DNA. Fibroblasts from out-bred neonatal mice and humans showed similar band-specific repair rate ratios and the global repair rate of murine fibroblasts was almost as rapid as that of the human fibroblasts. The mass distribution of the human Alu-probed signal was further analyzed. Gel mobility data was fitted to a logistic equation to include all M(r) values. Hypothetical distributions of DNA randomly cleaved to a particular number-average molecular weight were fit to the logistic gel mobility function to determine how such a randomly cleaved distribution of a particular cleavage frequency would be displayed along the experimental gel. This revealed a rapidly repaired kinetic fraction that represented 17% of the Alu-probed signal (R-band DNA), almost none of the L1 probed signal (G-band DNA), and reflects transcription coupled repair of active genes. The remaining Alu-probed DNA showed a random distribution of UVC-induced CPDs throughout all stages of global nucleotide excision repair. The Alu-probed CPDs disappeared with an excellent fit to first order kinetics and with a half-life of seven hours.
Sanders
M H
MH
Beckman Research Institute, Department of Biology, City of Hope Medical Center, Duarte, CA 91010, USA.
Bates
S E
SE
Wilbur
B S
BS
Holmquist
G P
GP
D020087:Alu Elements / Q000235:genetics / Q000528:radiation effects; D000818:Animals; D002478:Cells, Cultured / Q000378:metabolism; D002869:Chromosome Aberrations; D002871:Chromosome Banding; D003433:Crosses, Genetic; D004247:DNA / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair / Q000502:physiology*; D004276:DNA, Satellite / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D005347:Fibroblasts / Q000378:metabolism* / Q000528:radiation effects; D006801:Humans; D007231:Infant, Newborn; D007700:Kinetics; D020084:Long Interspersed Nucleotide Elements / Q000235:genetics / Q000528:radiation effects; D009115:Muridae; D011740:Pyrimidine Dimers / Q000378:metabolism*; D018626:Retroelements / Q000235:genetics* / Q000528:radiation effects; D020086:Short Interspersed Nucleotide Elements / Q000235:genetics / Q000528:radiation effects; D014466:Ultraviolet Rays
D004276:DNA, Satellite; D011740:Pyrimidine Dimers; D018626:Retroelements; D004247:DNA