Apoptosis induced by replication inhibitors in Chk1-depleted cells is dependent upon the helicase cofactor Cdc45.
Checkpoint kinase 1 (Chk1) responds to disruption of DNA replication to maintain the integrity of stalled forks, promote homologous recombination-mediated repair of replication fork lesions, and control inappropriate firing of replication origins. This response is essential for viability as replication inhibitors trigger apoptosis in S-phase cells depleted of Chk1. Given the complex network of cellular responses controlled by Chk1, our aim was to determine which of these protect cells from apoptosis following replication stress. Work with cell-free systems has shown that RPA-ssDNA complex forms following replication inhibition through the uncoupling of replication and helicase complexes. Here we show that replication protein A (RPA) foci form in cells treated with replication inhibitors and that the number of foci dramatically increases together with hyperphosphorylation of RPA34 in Chk1-depleted cells in advance of the induction of apoptosis. RPA foci, RPA34 hyperphosphorylation, and apoptosis were suppressed by siRNA-mediated knockdown of Cdc45, an essential replication helicase cofactor required for both the initiation and elongation steps of DNA replication. In contrast, loss of p21, a negative effector of origin firing, stimulates both the accumulation of RPA foci and apoptosis. Taken together, these results suggest that the loss of control of replication origin firing following Chk1 depletion triggers the accumulation of the RPA-ssDNA complex and apoptosis when replication is blocked.
Rodriguez
R
R
Institute for Cancer Studies, University of Sheffield, School of Medicine and Biomedical Sciences, Sheffield S10 2RX, UK.
Gagou
M E
ME
Meuth
M
M
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism
C113571:CDC45 protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004277:DNA, Single-Stranded; D034741:RNA, Small Interfering; D051716:Replication Protein A; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1