Abstract
Histone H2AX is phosphorylated at Ser139 in response to double-strand breaks (DSB) as well as stalled DNA replication forks and sometimes their collapse. We previously reported that histone H2AX is also phosphorylated in serum-starved quiescent cells exposed to UV in a nucleotide excision repair (NER)-dependent manner. We proposed a model that this phosphorylation is mediated by ATR, in response to single-stranded DNA (ssDNA) gaps, possibly caused by inefficient gap-filling reaction due to low cellular levels of repair replication factors, although ssDNA gap formation has not been experimentally proved.
In this study, we have tried to detect the ssDNA regions using immunostaining with anti-ssDNA antibody. Fluorescence signals in the nuclei were increased following UV exposure and further enhanced by the addition of Ara-C during post-UV incubation. Local UV irradiation clearly revealed that ssDNA formation is merged with H2AX phosphorylation. On the other hand, we also detected the accumulation of DSB-related factors including Nbs1, Mre11 and 53BP1 at locally UV-damaged subnuclear regions. In addition, we found that ATM Ser1981 and Chk2 Thr68 are phosphorylated following UV exposure under the serum-starved condition. Importantly, all reactions described above require cellular NER activity. These results suggest that UV-induced H2AX phosphorylation in G0-arrested human cells is mediated by ATR in response to ssDNA gap formation and also at least in part by ATM possibly activated by DSB formation, which are potentially generated by an NER process under the quiescent condition.
