Summary: Many chemotherapeutics kill cancer cells by inducing DNA damage interfering with DNA replication. Replication forks can reverse to aid the repair of DNA damage induced by chemotherapeutics, and BRCA proteins are key to protecting the reversed forks from nucleolytic degradation. In absence of BRCA, reversed replication forks are extensively degraded by nucleases, leading to chemosensitivity. Moreover, chemoresistance has been linked to the restored ability of BRCA-deficient cells to protect forks from degradation through mechanisms that remain unclear. Thus, understanding how cells protect stalled DNA replication forks and promote their recovery is critically important for developing and improving strategies for cancer therapy. In this application, we combine the expertise from the Zou and Vindigni laboratories to investigate the mechanisms of DNA replication fork protection and recovery in BRCA1-proficient (Aim 1) and -deficient (Aim 2) cells. The Zou lab has extensive experience in the ATR signaling pathway, which is crucial for stabilizing the genome during DNA replication. The Vindigni lab has discovered principal steps of the fork reversal pathway. Working together, we have uncovered a number of new players involved in fork protection. In the preliminary studies leading to Aim 1, we found that ATR plays a previously unrecognized role in protecting stalled/reversed replication forks from nucleolytic degradation in BRCA1-proficient cells. We also showed that ATR is required for the efficient recovery of stalled forks. Based on this premise, we hypothesize that ATR acts locally at stalled replication forks to protect reversed forks from nucleolytic degradation and to promote their restart after drug removal. Aim 1 will determine the mechanisms by which ATR protects stalled/reversed forks and promotes fork restart in BRCA1- proficient cells. In the preliminary studies leading to Aim 2, we have investigated how stalled forks are protected and how they recover in cells lacking BRCA1. We found that extensively degraded replication forks in BRCA1- deficient cells can recover through a pathway mediated by Rad18 and Ubc13. Furthermore, when BRCA1- deficient cells acquire PARP inhibitor resistance, fork protection is restored via a PALB2-dependent mechanism, which relies on ATR activity. Notably, both Ubc13 and PALB2 are functionally linked to the E3 ubiquitin ligase RNF168, raising the possibility that Ubc13, RNF168, and PALB2 may act in the same axis to protect stalled forks and promote fork recovery independently of BRCA1. We hypothesize that both Ubc13 and PALB2 have unanticipated roles in fork recovery/protection in the absence of BRCA1, and their functions may be linked by RNF168. Aim 2 will investigate how Ubc13 promotes fork rec...