Eukaryotic genome replication is surveyed by the S-phase checkpoint, which coordinates sequential origin activation to prevent the exhaustion of poorly defined, rate-limiting replisome components1-3. Here we show that excessive origin firing saturates chromatin-bound proliferating cell nuclear antigen (PCNA)-a sliding clamp for DNA polymerase processivity and Okazaki fragment processing4-thereby restricting further PCNA loading and lagging-strand synthesis when checkpoint control is lost. PCNA-associated factor 15 (PAF15) emerges as a dosage-sensitive regulator of this process5-9. During unperturbed S phase, the entire soluble PAF15 pool binds to chromatin, leaving no reserve to stabilize PCNA under conditions of excessive origin activation. PAF15 binds to PCNA specifically on the lagging strand through a high-affinity PIP motif and occupies the DNA-encircling channel, protecting the clamp and associated enzymes from premature unloading by the ATAD5-RFC complex. Conversely, overexpression of PAF15 or forced redistribution to the leading strand disrupts replisome progression and induces cell death. These detrimental effects are mitigated by Timeless-Claspin, which blocks PAF15-PCNA binding on the leading strand. E2F4-mediated repression fine-tunes PAF15 expression to ensure optimal dosage and strand specificity. These findings reveal a previously unrecognized replisome constraint: when PAF15-PCNA assemblies are exhausted, the S-phase checkpoint globally restricts origin activation, linking a strand-specific rate-limiting mechanism to global replication dynamics.

Center for Functional Genomics and Tissue Plasticity University of Southern Denmark Odense Denmark

Department of Biochemistry and Molecular Biology University of Southern Denmark Odense Denmark

Department of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic

Genome Integrity and Cancers Gustave Roussy Université Paris Saclay CNRS Villejuif France

Institute of Biophysics of the Czech Academy of Sciences Brno Czech Republic

Novo Nordisk Foundation Center for Genomic Mechanisms of Disease Broad Institute of MIT and Harvard Cambridge USA

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