Dynamic regulation of origin firing factors links CDK activity to dormant origin activation

Eukaryotic cells license far more potential replication origins than they use, but how origin firing rate is tuned in S phase, especially under replication stress, remains unclear. Here, we identify a regulatory mechanism by which cyclin-dependent kinase (CDK) activity controls the abundance and chromatin recruitment of the origin firing factors TRESLIN and MTBP to promote dormant origin activation. Inhibition of WEE1 kinase during S phase increases CDK activity, which blocks the PCNA-CRL4^CDT2-dependent degradation of TRESLIN and enhances its chromatin association along with MTBP. This increased loading is required for elevated helicase recruitment and DNA synthesis under CDK-hyperactive conditions. We define a sequence within TRESLIN required for its degradation and show that TRESLIN stabilization is necessary but not sufficient for CDK-driven origin firing. Replication stress regulates TRESLIN destruction in a dose- and time-dependent manner. Acute high-dose hydroxyurea suppresses CDK activity and reduces chromatin-bound TRESLIN, whereas prolonged low-dose hydroxyurea is accompanied by elevated CDK activity and increased chromatin loading of TRESLIN-MTBP and CDC45. Altogether, these findings uncover a control point in replication origin usage with implications for genome stability and therapeutic kinase inhibition.