Greater than 50% of advanced melanoma patients develop resistance or fail to respond to current standard-of-care treatments, including targeted therapies (e.g., BRAF/MEK inhibitors) and immunotherapies (e.g., anti-PD1, anti-CTLA4). The Gabrielli laboratory has developed a novel tumour-selective combination therapy combining a checkpoint kinase 1 inhibitor (CHK1i) with low-dose hydroxyurea (LDHU) to exploit the endogenous replication stress (RS) commonly observed in melanoma. We propose that CHK1i+LDHU intensifies RS during S- phase by promoting unscheduled origin firing, disrupting the spatiotemporal regulation of DNA replication, and generating excessive single-stranded DNA (ssDNA). This ssDNA may exceed the buffering capacity of replication protein A (RPA), leaving it vulnerable to nucleolytic degradation, consistent with the ‘RPA Exhaustion Model’ of DNA damage. In this study it is demonstrated that CHK1i+LDHU induced S-phase re-ordering with persistent early- replicating topologically associated domains (TADs) and reduced late-replicating lamina- associated domains (LADs), consistent with unscheduled origin firing. Replication fork activity was reduced, while pan-nuclear accumulation of ssDNA and γH2AX emerged as key features. High-content imaging showed ssDNA and γH2AX did not correlate at the single-cell level, suggesting distinct temporal or mechanistic pathways. γH2AX strongly correlated with pATM S1981, and ATM inhibition reduced γH2AX in two of three cell lines. Notably, pan- nuclear pATM S1981 staining was the predominant phenotype in CHK1i+LDHU-treated cells, contrasting with the commonly reported focal pATM response. We speculate that CHK1i+LDHU promotes chromatin relaxation, facilitating ATM recruitment via 53BP1 and enhancing BrdU antibody accessibility, and the pan-nuclear γH2AX observed. These findings suggest that CHK1i+LDHU induces nuclear-wide ATM activation and gH2AX phosphorylation through unscheduled origin firing and ssDNA accumulation. Whether this leads to RPA exhaustion remains to be tested. Importantly, our results indicate that CHK1i+LDHU exploits cancer cells with endogenous RS like melanoma, offering a novel therapeutic strategy.