Dissecting the functional relationship between RAD54L and FBH1 in replication fork reversal

About This Grant

Project Summary This proposal outlines a training program for an aspiring principal investigator with a focus on developing a comprehensive skillset to make impactful discoveries in our understanding of the mechanisms that overcome replication stress. Replication stress, the slowing and stalling of DNA synthesis, endangers accurate DNA replication and threatens genome stability. Replication stress is induced in healthy cells through various endogenous and exogenous factors, such as a shortage in deoxyribonucleotides or exposure to reactive oxygen species. In genotoxic cancer therapy, however, replication stress in tumor cells is anticipated, and pathways that mitigate replication stress lead to resistance to treatment and to recurrence of disease. Consequently, there is a critical need to understand the molecular mechanisms by which both healthy and cancerous cells overcome replication stress. The proposed project will impact human health by uncovering the molecular mechanisms of proteins that mitigate replication stress through their engagement in replication fork (RF) reversal. In RF reversal, the RF undergoes a structural change that involves the annealing of the nascent and parental DNA strands to drive the movement of the RF in the reverse direction. In the reversed configuration, the RF is stabilized and able to withstand loss of its integrity from replication stress through repair and checkpoint mechanisms. In human cells, two distinct RF reversal pathways have been described- one relying on SMARCAL1 with HLTF and ZRANB3 and the other on FBH1. However, the molecular mechanisms of the FBH1-pathway of RF reversal have remained unclear, leaving a significant knowledge gap in the field. As such, there is a critical need to delineate the molecular details of FBH1-mediated RF reversal. The central hypothesis of this project is that RAD54L, a critical protein in DNA repair by homologous recombination, is essential in the FBH1-mediated pathway of RF reversal. Our overall objectives are to explicate the molecular mechanisms of FBH1 and RAD54L in RF reversal in human cells (Aim 1), determine the physical and functional interaction between RAD54L and FBH1 in vitro (Aim 1), and investigate the consequences of impaired RF reversal in pre-neoplastic cells with an activated oncogene (Aim 2). The proposed research is significant because it is expected to provide scientific justification for the continued development of inhibitors targeting pathways that mitigate replication stress. The knowledge gained herein also will improve risk predictions for health of individuals with altered genes and proteins critical in RF reversal. The proposed research will incorporate expert training in cell biology, biochemistry, and model organisms in the F99 and K00 phases. The F99 phase will be completed at Colorado State University under the mentorship of Dr. Claudia Wiese with additional biochemical training from Dr. Patrick Sung at the University of Texas Health Science Center San Antonio. The K00 phase will be completed at a separate institution under the K00 mentor.