PTBP1 enforces ATR-CHK1 signaling determining the potency of CDC7 inhibitors.

CDC7 kinase is crucial for DNA replication initiation and fork processing. CDC7 inhibition mildly activates the ATR pathway, which further limits origin firing; however, to date the relationship between CDC7 and ATR remains controversial. We show that CDC7 and ATR inhibitors are either synergistic or antagonistic depending on the degree of inhibition of each individual kinase. We find that Polypyrimidine Tract Binding Protein 1 (PTBP1) is important for ATR activity in response to CDC7 inhibition and genotoxic agents. Compromised PTBP1 expression makes cells defective in RPA recruitment, genomically unstable, and resistant to CDC7 inhibitors. PTBP1 deficiency affects the expression and splicing of many genes indicating a multifactorial impact on drug response. We find that an exon skipping event in RAD51AP1 contributes to checkpoint deficiency in PTBP1-deficient cells. These results identify PTBP1 as a key factor in replication stress response and define how ATR activity modulates the activity of CDC7 inhibitors.