Strand-independent degradation of uncoupled forks by EXO1 activates ATR and restrains fork progression

Abstract

Nascent DNA degradation of replication forks shapes genome stability, yet the mechanism of degradation, and its direct consequences, remain unclear. Here we use localized nascent strand degradation at replication forks in Xenopus egg extracts to examine the mechanism and consequences of degradation. The exonuclease EXO1 is crucial for degradation and acts specifically on replication fork structures. Degradation requires EXO1 catalytic activity and is defective in the E109K Lynch Syndrome associated mutant. EXO1 degrades both nascent strands 5-3: the lagging strand from its native 5 end and the leading strand from a distal 5 entry on the sister fork, whereas leading strand 3 end remains stable. Impaired leading-strand degradation at a specific site does not affect degradation of the corresponding lagging-strand region, so the two strands are degraded independently. Degradation of the uncoupled fork has two downstream consequences. It is important to activate ATR, which is otherwise weakly activated by the uncoupled fork. Additionally, degradation restrains fork progression, independent of ATR activation. Our findings demonstrate that strand-independent degradation of uncoupled forks by EXO1 activates ATR and restrains fork progression