Two fork protection complexes at the replication fork play distinct roles in fork progression and stress response.

TIMELESS, together with TIPIN and CLASPIN, forms the Fork Protection Complex (FPC), an essential regulator of DNA replication that possesses multiple functions in genome stability including the regulation of fork progression and replication checkpoint signaling. Structural studies place TIMELESS at the leading edge of the CMG helicase, which is inconsistent with FPC functions at the lagging strand and on single-stranded DNA. Our observation that FPC chromatin loading during replication initiation started in G1 phase cells, but was also enhanced by DNA synthesis, led us to propose a model of a step-wise loading of the FPC with two TIMELESS molecules per replication fork. Split-TurboID proximity labelling supported this model, placing the second FPC in proximity to the lagging strand. Using an auxin-inducible degron, we show that TIMELESS depletion compromised chromatin loading of TIPIN and CLASPIN, but the TIMELESS mutant unable to bind MCM still supported CLASPIN and TIPIN chromatin loading. This mutant was proficient in replication checkpoint activation, but failed to regulate fork speed under both unperturbed and oxidative-stress conditions. We propose that two distinct FPC instances at each replication fork: one at the leading edge, regulating fork progression, and one at the lagging strand mediating checkpoint signaling, - together execute the essential functions of TIMELESS.