Completion of excision repair in human cells. Relationship between ligation and nucleosome formation

The completion of excision repair patches in human cells, following UV irradiation, was compared to the refolding of these regions into nucleosomes. Incomplete repair patches were detected by their enhanced sensitivity to exonuclease III. This enhanced sensitivity was due to the presence of gaps (or displaced parental strands) at the 3' end rather than unligated nicks, indicating that ligation occurs rapidly after repair synthesis is completed. Different rates of completion were achieved by treatment with the inhibitors hydroxyurea and sodium butyrate, as well as by using a (partially) ligase-deficient human cell strain. Hydroxyurea caused a marked decrease in both the rate of completion and the level of repair incorporation in all three cell types studied, while sodium butyrate yielded different effects in each cell type. In each case, however, a decrease in the rate of repair patch completion resulted in a concomitant decrease in the level of nucleosome formation. To determine the temporal relationship of these two events, the levels of repair-incorporated nucleotides in isolated 146-base pair nucleosome core DNA were compared on native and denaturing gels. The data indicate that little (or no) nucleosome formation occurred in the nascent DNA regions prior to ligation regardless of the cell type or treatment used. Furthermore, comparison of the fraction of unligated repair patches and the fraction of repair patches in a nonnucleosomal state indicated that in the absence of inhibitors there was a significant time lag between ligation and nucleosome formation. This lag time, however, decreased when cells were treated with hydroxyurea. Thus, the formation of nucleosomes in newly repaired regions of DNA occurred after the ligation step in all cases and these two features of the excision repair process are not "tightly coupled" events.