Multiple conformational states of repair patches in chromatin during DNA excision repair

In mammalian cells, newly synthesized DNA repair patches are highly sensitive to digestion by staphylococcal nuclease (SN), but with time, they acquire approximately the same nuclease resistance as the DNA in bulk chromatin. We refer to the process which restores native SN sensitivity to repaired DNA as chromatin rearrangement. We find that during repair of ultraviolet damage in human fibroblasts, repair patch synthesis and ligation occur at approximately the same rate, with ligation delayed by about 4 min, but that chromatin rearrangement is only 75% as rapid. Thus, repair-incorporated nucleotides can exist in at least three distinct states: unligated/unrearranged, ligated/unrearranged, and ligated/rearranged. Inhibition of repair patch synthesis by aphidicolin or hydroxyurea results in inhibition of both patch ligation and chromatin rearrangement, confirming that repair patch completion and/or ligation are prerequisites for rearrangement. We also analyze the kinetics of SN digestion of repair-incorporated nucleotides at various extents of rearrangement and find the data to be consistent with the existence of two or more forms of unrearranged repair patch which have different sensitivities to digestion by SN. These data indicate that the chromatin rearrangement which restores native SN sensitivity to repaired DNA is a multistep process. The multiple forms of unrearranged chromatin with different SN sensitivities may include the unligated/unrearranged and ligated/unrearranged states. If so, the differences in SN sensitivity must arise from differences in chromatin structure, because SN does not differentiate between ligated and unligated repair patches in naked DNA.