Analysis of the Replication Stress Response

PROJECT SUMMARY Billions of base pairs of DNA must be replicated trillions of times during a human lifetime. Adding to the difficulty, replication is challenged by stresses including DNA template lesions, difficult to replicate sequences, and conflicts with transcription. Multiple repair and tolerance responses to replication stress act to complete DNA synthesis while minimizing errors. In this project we seek to understand these mechanisms with a focus on replication fork reversal. Replication fork reversal is thought to stabilize stalled forks, to place DNA lesions back into the context of duplex DNA where it can be removed through excision repair, and to provide an opportunity for template switching. Reversal is catalyzed by ATP-dependent DNA translocases and the RAD51 recombinase. It is also highly regulated, which is needed to prevent inappropriate slowing of replication and nuclease- dependent processing of replication forks. For example, we recently discovered RADX as a single-stranded DNA binding protein that regulates reversal by directly binding RAD51. In this funding period we seek to understand how RADX functions, how replication resumes following a challenge, and to rigorously test the hypothesis that fork reversal is an error-free mechanism of DNA damage tolerance. We will test and refine new conceptual models utilizing a combination of biochemical, genetic, and cell biological approaches. Our many years of accumulated knowledge and reagents and strong team of collaborators uniquely position us to complete these studies. Given the importance of replication stress responses to preventing diseases including cancer, these studies will generate discoveries that could be translated in the future to benefit human health.