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

Abstract The degradation of nascent DNA at stalled replication forks is critical for genome integrity, yet the specific mechanisms of degradation at uncoupled forks and the resulting functional consequences remain poorly understood. We induced site-specific replication fork uncoupling using Xenopus egg extracts in order to examine how degradation affected the different DNA structures formed, compare degradation of leading and lagging strands, and interrogate the resulting functional consequences. We found that EXO1 is critically important for degradation of uncoupled forks, independent of any degradation at reversed forks. Lagging strands are rapidly degraded from their native 5’ end by EXO1, while the nascent leading strand 3’ end is not detectably degraded. Sequences distal to the leading 3’ end are also degraded by EXO1 due to degradation arising from the lagging strand of the diverging sister fork. Importantly, impaired leading strand degradation does not impact lagging strand degradation at the same locus, indicating that degradation of the two strands is independent. Degradation by EXO1 has two major functional consequences: it is required to generate the signal that activates the ATR checkpoint at uncoupled forks; and it restrains fork progression. Overall, our results show that replication fork uncoupling causes degradation of 5’ ends that is crucial for ATR signaling and fork slowing, while 3’ ends are highly stable.