Xuejiao Yang:
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DNA double-strand breaks (DSBs) are the most deleterious form of damaged DNA in cells of both prokaryotes and eukaryotes, since an irreparable DSB can result in cell death. In the budding yeast Saccharomyces cerevisiae (S. cerevisiae), homologous recombination (HR) is a main DSB repair pathway, especially in S- and G2-phase cells. In the HR, an important step at the early stage is the formation of replication protein A (RPA)-coated single-stranded DNA (ssDNA), which can recruit DNA damage checkpoint proteins such as Mec1-Lcd1 (Ddc2) complex. The recombinase Rad51 is a key factor in HR repair, which forms a Rad51 nucleoprotein filament by replacing RPA from ssDNA, catalyzing homology search and strand exchange during the HR. In order to learn more about the regulation of Rad51 in this pathway, I performed a genome-wide BiFC-based proteinprotein interaction screening for Rad51, to map the interaction network of Rad51 in living cells of S. cerevisiae. Intriguingly, some checkpoint-related interactions of Rad51 were identified by this screen, and the Rad51-Lcd1 interaction attracted my most attention. I proposed a hypothesis for the interaction of Rad51-Lcd1 that Rad51 is involved in activating DNA damage checkpoint after replacing RPA from ssDNA, relying on its interaction with Lcd1. The alterations of Rad53 phosphorylation (detected by western blot) and autophosphorylation (detected by kinase assay) levels in wild-type and rad51Δ cells with or without Rfa1 depletion indicate that Rad51 indeed contributes to promote and maintain DNA damage checkpoint activation.