DNA2 drives processing and restart of reversed replication forks in human cells

TLDR

Accurate processing of stalled or damaged DNA replication forks is essential for genomic integrity, with recent work highlighting fork reversal and restart as central mechanisms for high‑fidelity replication. The study identifies a novel DNA2‑ and WRN‑dependent mechanism for processing and restarting reversed replication forks after prolonged genotoxic stress. The mechanism involves DNA2 nuclease and WRN ATPase activities that degrade reversed forks with 5′‑to‑3′ polarity to promote replication restart. The study shows that DNA2 and WRN cooperate to 5′‑to‑3′ degrade reversed forks and restart replication, while EXO1, MRE11, and CtIP are not involved, RECQ1 restrains DNA2 to prevent excessive nascent strand loss, and RAD51 depletion blocks fork reversal, collectively defining a new, tightly regulated mechanism for genome integrity.

Abstract

Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5'-to-3' polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors.