Abstract B017: VCP extracts the chromatin remodeler SNF2H from nascent DNA to stabilize stressed replication forks

Abstract

Abstract Valosin-containing protein (VCP) is an evolutionarily conserved AAA+ ATPase which plays pleiotropic roles in global proteostasis by extracting polyubiquitinated proteins from various cellular organelles and structures and facilitating their turnover. Our recent work demonstrated that nuclear VCP activity during DNA damage response specifically depends on ATM/ATR/DNA-PK-mediated Ser784 phosphorylation. Ser784phosphorylation increases VCP ability to facilitate chromatin-associated protein degradation and is required for DNA repair, checkpoint signaling, and cell survival. Clinically, high nuclear pSer784-VCP levels are significantly associated with poor survival outcome among chemotherapy-treated breast and pancreatic cancer patients. Thus, pSer784-VCP is a broadly important and therapeutically relevant genome stabilizer. Here, we focused specifically on the role of pSer784-VCP in DNA replication fork stability during stress, a known source of DNA damage if not properly regulated. By performing single-molecule DNA fiber analysis using human cell lines with genetic gain and loss-of-function of VCP, we observed that VCP protects nascent DNA from hydroxyurea (HU)-induced over-resection and this activity requires Ser784 phosphorylation. Using proximity ligation assay (PLA), we detected HU-induced pSer784-VCP on nascent DNA, suggesting that it may protect stressed forks by extracting functionally important regulators. Consistent with this theory, we detected an interaction of pSer784-VCP with SNF2H, a nucleosome-sliding enzyme which we recently found essential for nascent DNA over-resection at unprotected forks. In support of the hypothesis that SNF2H is a novel substrate of pSer784-VCP at stressed forks, we observed SNF2H retention on nascent DNA upon VCP knockdown in HU-treated cells, and the functional rescue of SNF2H retention by VCP re-expression requires Ser784phosphorylation. In support of their epistatic relationship, SNF2H depletion fully rescued fork over-resection caused by VCP loss. Similarly, blocking fork reversal by depleting SMARCAL1 or FBH1 also rescued fork over-resection caused by VCP loss. Importantly, mutating the ATPase activity of SNF2H abolished its ability to promote fork over-resection, suggesting that nucleosome remodeling by SNF2H underlies its effect on fork stability and may be a key molecular event enabling fork reversal upstream of fork degradation. Collectively, our data suggest that pSer784-VCP is a previously unrecognized stabilizer of stressed DNA replication forks, and it does so at least in part via its physical extraction of SNF2H from nascent DNA and the consequent attenuation of nucleosome remodeling needed for fork reversal and over-resection. More experiments are currently underway to further dissect the mechanistic aspects of this working model as well as to evaluate the potential of developing pSer784-VCP into a clinically relevant chemo-predictive biomarker and chemo-sensitizing target for different types of cancer. Citation Format: Jieya Shao, Mari Iwase, Rong Xu, Shuyang Lin. VCP extracts the chromatin remodeler SNF2H from nascent DNA to stabilize stressed replication forks [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B017.