Our laboratory has developed phospho-specific BLM helicase (T99p-BLM) antibodies to study DNA damage induced by camptothecins and topoisomerase I (Top1) inhibitors. Top1 is the selective molecular target of camptothecins that act by trapping Top1-associated DNA single-strand breaks (SSB). These SSB are lethal after being converted to double-strand breaks by replication forks as they reach the Top1-mediated SSB. The resolution of stalled replication forks (Holliday junctions) is critical for genomic stability. BLM, a RecQ DNA helicase, and topoisomerase-IIIα (Top3α) appear essential to resolve stalled replication forks. Targeting Top1 leads to BLM phosphorylation on T99 and altered the nuclear localization pattern of the phosphorylated form of BLM. T99 phosphorylated BLM displays reduced co-localization with Top3α and complete co-localization with γ-H2AX, a known marker of DNA double strand breaks. Replication-dependent assembly of a multi-protein complex in response to camptothecin consisting of the phosphorylated forms of ATM, Chk2, BLM and H2A is demonstrated using confocal imaging. Using genetically modified cell lines, we identified ATM and ATR as the kinases responsible for phosphorylating BLM on T99. In response to camptothecin, BLM-deficient cells exhibit higher levels of Top1-DNA complexes than BLM-proficient cells. Such an increase of unrepaired DNA damaging lesions might, at least in part, explain the camptothecin-sensitivity in BLM-deficient cells. However, BLM-deficient cells had reduced formation of γ-H2AX foci. Mechanistically, the resolution of stalled replication forks and facilitation of γ-H2AX activation by BLM helicase might facilitate repair of the replication lesions caused by Top1-trapping. Our studies raise the possibility that cancer cells defective for BLM might be selectively sensitive to camptothecins. T99 phosphorylated BLM might serve as a useful therapeutic biomarker for Top1 inhibitors including camptothecins and novel inhibitors such as indenoisoquinolines. We have also begun to extend our studies using other DNA and non-DNA targeting anti-cancer agents to further understand the mechanistic importance of phosphorylating BLM.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]