Correlation between repair patch ligation and nucleosome rearrangement in human cells treated with bleomycin, UV radiation or methyl methanesulfonate.
We have examined ligation and nucleosome rearrangement of repair patches in chromatin of human fibroblasts damaged with bleomycin (BLM), UV radiation and methyl methanesulfonate (MMS) to follow completion of excision repair involving different combinations of repair enzymes. Conditions were used that allowed analysis of the correlation between these two events over a large range (i.e. from 5% to greater than 99% ligated). Cells exposed to BLM were reversibly permeabilized with L-alpha-lysophosphatidylcholine and pulse-labeled with either [3H]dTTP or [3H]dThd to label selectively cells that 'reseal their membranes' at different rates. A striking difference is observed in the rates of ligation of these nascent repair patches, in that those labeled with [3H]dTTP are ligated much slower (25-50% unligated after 24 h) than those labeled with [3H]dThd (less than 5% unligated after 6 h). The nuclease sensitivity of [3H]dTTP-labeled patches also decreases more slowly, indicating that the rate of nucleosome rearrangement decreases compared to that of repair patches labeled with [3H]dThd. The rates of repair patch ligation and loss of nuclease sensitivity were also modulated in intact cells exposed to UV radiation or MMS by treatment with aphidicolin and/or hydroxyurea. A plot of relative nuclease sensitivity versus fraction of ligated repair patches yields an overall linear correlation of greater than 0.8 in each case, indicating that these two features of nascent repair patches are 'moderately coupled' events. These results support the idea that ligation of repair patches is a prerequisite for nucleosome rearrangement following three different modes of excision repair.
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660.
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Smerdon
D016590:Aphidicolin / Q000494:pharmacology; D001761:Bleomycin / Q000494:pharmacology*; D002478:Cells, Cultured; D004260:DNA Repair; D006801:Humans; D008741:Methyl Methanesulfonate / Q000494:pharmacology*; D009707:Nucleosomes / Q000187:drug effects* / Q000528:radiation effects; D013942:Thymine Nucleotides / Q000378:metabolism; D014466:Ultraviolet Rays
D009707:Nucleosomes; D013942:Thymine Nucleotides; D001761:Bleomycin; D016590:Aphidicolin; D008741:Methyl Methanesulfonate; C024157:thymidine 5'-triphosphate