PAF15-PCNA assembly exhaustion governs lagging strand replication and replisome integrity

Abstract

Genome replication in eukaryotic cells is surveyed by the S-phase checkpoint, which orchestrates sequential replication origin activation to avoid exhaustion of hitherto poorly defined rate-limiting replisome components. Here, we find that excessive activation of replication origins depletes chromatin-bound PCNA and lagging strand components, thereby limiting additional PCNA loading at new origins when checkpoint control is disrupted. PAF15 (PCNA-associated factor 15) emerges as a dosage-sensitive regulator of PCNA, delineating the dynamic range of global genome duplication and defining distinct roles for PCNA on the leading and lagging strands. Through its high-affinity PIP motif and interaction within the DNA encircling channel of PCNA, PAF15 stabilizes PCNA exclusively on the lagging strand, optimizing and rate-limiting lagging strand processing. On the other hand, misregulation of PAF15--whether by overexpression or mislocalization to the leading strand--impairs replication fork progression and leads to cell death. These defects are mitigated by TIMELESS and CLASPIN, which restrain PAF15-PCNA interactions beyond the lagging strand. E2F4-mediated repression orchestrates PAF15 expression in normal and cancer cells, maintaining its optimal dosage for lagging strand-specific interactions with PCNA. Thus, the S-phase checkpoint functions in concert to restrict origin activation when lagging strand PAF15-PCNA assembly is exhausted, linking a previously concealed strand-specific rate limitation to overall replication dynamics.