ATM promotes nucleolytic processing of reversed replication forks during DNA interstrand cross-link repair

During replication-coupled DNA interstrand cross-link (ICL) repair, fork reversal is thought to enable the Fanconi anemia (FA) pathway to resolve the ICL through nucleolytic incisions. The resulting regressed arm of the reversed fork undergoes extensive nucleolytic resection that may facilitate completion of replication. Despite the importance of these remodeling events, their regulation remains poorly understood. Here, we use cell-free Xenopus egg extracts and human cells to investigate reversed fork processing during ICL repair by the FA pathway. We find that the ataxia-telangiectasia mutated (ATM) kinase is activated concomitantly with fork reversal and promotes resection of reversed fork intermediates. ATM enhances chromatin recruitment of DNA2, which acts alongside EXO1 to resect reversed forks formed during ICL repair. Moreover, inhibition of protein phosphatase 2A (PP2A) leads to ATM hyperactivation, accelerated reversed fork processing, and accumulation of aberrant end-joining products, implying that PP2A restrains ATM signaling to limit reversed fork resection. Taken together, this work identifies reversed forks as substrates for ATM activation and reveals a phospho-regulatory circuit that controls reversed fork processing. Replication stress often induces fork reversal, which protects stalled replication intermediates until replication can resume. This study identifies the ATM kinase as a key regulator that promotes nuclease-dependent processing of reversed fork structures formed at DNA interstrand cross-links.