CDC7 kinase regulates DNA replication fork processing and its inhibition is modulated by PTBP1
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CDC7 is an essential serine/threonine kinase that regulates the initiation of DNA replication by phosphorylating several sites of the MCM helicase complex. Several inhibitors of CDC7 are available that reduce cell proliferation by limiting DNA replication initiation. As these inhibitors are in development as anti-cancer therapeutics, it is crucial to identify the additional roles of CDC7 and understand cellular response to CDC7 inhibitors. Novel evidence has implicated CDC7 in the cellular response to replication stress. Replication stress can be caused by a variety of endogenous and exogenous insults that impair replication progression. It is characterised by fork slowing and arrest. One type of DNA intermediate that can form in response to replication stress is a reversed fork. This four-branched structure results from strand exchange reactions in which the nascent strands anneal and the parental strands re-anneal. Replication fork reversal maintains replication integrity and allows time for the stress or obstacle to be overcome before replication can restart. Prolonged fork stalling or incorrect processing can lead to fork collapse and genome instability, a hallmark of cancer. Using chemical inhibitors of CDC7 we have demonstrated that CDC7 activity is required for replication fork processing and a robust signalling response upon replication stress. Using biochemical approaches we find that CDC7 localises to replication forks and, along with MRE11, is retained at stalled forks in a manner dependent on CDC7 activity. We also find that MRE11 is phosphorylated in response to replication stress, and this is attenuated upon CDC7 inhibition. Importantly, we find that CDC7 promotes MRE11 dependent fork degradation in BRCA2 deficient cells. Therefore identifying CDC7 as a key modulator of replication initiation and elongation. To identify the genes that control the cellular response to CDC7 inhibition, a loss of function genome wide screen was performed in our laboratory. It has identified PTBP1 as a gene that restrains proliferation when CDC7 is inhibited. PTBP1 has roles in mRNA metabolism. It canonically functions to regulate exon usage, but also influences mRNA stability, translation and post transcriptional modifications. We use CRISPR/Cas9 technology to create a PTBP1 null cell line and a heterozygous mutant cell line. Using these cell lines we demonstrate that loss or partial depletion of PTBP1 allows cells to proliferate and have efficient DNA synthesis under conditions of limited CDC7 activity. Using RNA-seq analysis we show that loss of PTBP1 results in a drastic change in the transcriptome and a differential response to CDC7 inhibition. Interestingly, we show that PTBP1 mutated cells have attenuated checkpoint response to CDC7 inhibition and DNA alkylating agents, suggesting that PTBP1 is important for modulating the checkpoint response. These results show that expression levels of PTBP1 is a key determinant of cell sensitivity to CDC7 inhibitors.