Nucleosome rearrangement in human cells following short patch repair of DNA damaged by bleomycin.
We have examined the structure of newly repaired regions of chromatin in intact and permeabilized human cells following exposure to bleomycin (BLM). The average repair patch size (in permeabilized cells) was six to nine bases, following doses of 1-25 micrograms/mL BLM, and greater than 80% of the total repair synthesis was resistant to aphidicolin. In both intact and permeabilized cells, nascent repair patches were initially very sensitive to staphylococcal nuclease, analogous to repair induced by "long patch" agents, and are nearly absent from isolated nucleosome core DNA. Unlike long patch repair, however, the loss of nuclease sensitivity during subsequent chase periods was very slow in intact cells, or in permeabilized cells treated with a low dose of BLM (1 microgram/mL), and was abolished by treatment with hydroxyurea (HU) or aphidicolin (APC). The rate of repair patch ligation did not correlate with this slow rate of chromatin rearrangement since greater than 95% of the patches were ligated within 6 h after incorporation (even in the presence of HU or APC). In permeabilized cells, repair patches induced by either 5 or 25 micrograms/mL BLM, where significant levels of strand breaks occur in compact regions of chromatin, lost the enhanced nuclease sensitivity at a rate similar to that observed following long patch repair. This rapid rate of rearrangement was not affected by APC. These results indicate that short patch repair in linker regions of nucleosomes, and/or "open" regions of chromatin, involves much less nucleosome rearrangement than long patch repair or short patch repair in condensed chromatin domains.
Sidik
K
K
Biochemistry/Biophysics Program, Washington State University, Pullman 99164-4660.
Smerdon
M J
MJ
D000477:Alkylating Agents / Q000494:pharmacology; D001761:Bleomycin / Q000494:pharmacology*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D002843:Chromatin / Q000502:physiology; D004247:DNA / Q000187:drug effects* / Q000528:radiation effects; D004249:DNA Damage; D004256:DNA Polymerase I / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication / Q000187:drug effects; D006801:Humans; D009154:Mutation; D009707:Nucleosomes / Q000187:drug effects* / Q000528:radiation effects; D012680:Sensitivity and Specificity; D014466:Ultraviolet Rays
D000477:Alkylating Agents; D002843:Chromatin; D009707:Nucleosomes; D001761:Bleomycin; D004247:DNA; D004256:DNA Polymerase I