Understanding the regulatory mechanisms for Replication Termination Factor 2 (RTF2) removal and function during DNA replication

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Project Summary/ Abstract DNA replication is a highly regulated process that occurs in the physical arena of chromatin. The DNA replication machinery organizing this process, the replisome, confronts many obstacles, both endogenous and exogenous, that threaten the accurate and efficient duplication of the genome. Cells have multiple mechanisms to respond to these challenges and bypass lesions. Still, DNA replication stress has emerged as a potent source of endogenous damage driving genome instability and cancer. Our laboratory has identified a novel replication stress response axis in which the proteasome shuttle proteins DNA Damage Inducible 1 (DDI1) and DNA Damage Inducible 2 (DDI2), function to remove Replication Termination Factor 2 (RTF2) from stalled forks to mediate a proper recovery. Conversely, depletion of DDI1/2 results in retention of RTF2 in the replisome which causes a replication fork restart defect that leads to severe genomic instability and cell death. The observed cell death upon DDI1/2 depletion can be rescued by reducing levels of RTF2 at the replication fork. Overall, our laboratory has therefore shown that the replisome is reorganized during the replication stress response by the regulated removal of an essential replisome component. In this study, we will elucidate the mechanism by which RTF2 is removed from the replication fork and determine where RTF2 functions are required in the genome to maintain genome stability. Given that RTF2 is removed by a proteosome shuttle protein, we hypothesize that the removal of RTF2 requires the ubiquitin proteasome system. Both ubiquitin conjugation to RTF2 and degradation of the ubiquitin tagged RTF2 must be tightly regulated and efficient. We have already shown that DDI1/2 regulates the actual removal, but the mechanism of recognition of RTF2 by DDI1/2 needs to be understood. We propose that DDI1/2 interacts with a ubiquitin-conjugation on RTF2 to target its degradation and aim to identify (1) the specific residue(s) on RTF2 that must be ubiquitinated for recognition by DDI1/2 and (2) the targeting E3 ligase that ubiquitinates RTF2. Furthermore, we strive to understand the locations throughout the genome that require the prompt removal of RTF2 and define the chromatin niche wherein RTF2 functions. Chromatin is an ordered, yet dynamic regulatory platform composed of DNA and histone proteins and functionally distinct chromatin environments are created, in part, through reversible covalent post-translational modifications (PTMs) of histones. Our goal is to determine the genomic locations and chromatin environment that require RTF2 function and identify genomic loci that require RTF2 removal. With this project, we are studying the fundamental events that coordinate regulation between the ubiquitin proteosome system (UPS), chromatin, and the replisome at active and stalled replication forks to promote genome stability.