Mechanisms of replication fork repair

Vanderbilt University, Nashville TN

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Abstract

? DESCRIPTION (provided by applicant): Accurate and timely DNA replication is critical for survival and proper function of the cell, but takes place amidst an array of DNA lesions that impede progression of the replication fork. Stalled replication forks are a significant threat to genome stability that can lead to genome rearrangements or cell death. Replication fork reversal represents one DNA damage tolerance (DDT) pathway important for repair and restart of stalled forks, yet we know very little about the mechanisms of fork reversal. SMARCAL1 and HLTF enzymes are central components of the DDT response that preserve genome integrity through fork reversal. Mutations in smarcal1 cause Schimke Immunoosseous Dysplasia (SIOD), a disorder that usually results in death before the age of 15. HLTF plays a role in tumor suppression in human cells, and loss of HLTF expression is seen in various cancer types. Thus, it is important to human health that we develop a clearer understanding of the SMARCAL1 and HLTF proteins and their roles in DDT. SMARCAL1 and HLTF contain a unique ATP-dependent DNA annealing activity that promotes reversal and branch migration of model replication forks in vitro and that in SMARCAL1 is essential for prevention of fork collapse in vivo. The long-term goal of this research is to understand the mechanisms by which fork reversal prevents the demise of stalled replication forks in the cell. Here, we propose to determine the mechanisms of fork remodeling activities by SMARCAL1 and HLTF using a combination of structural biology, biochemistry, and cell biology. Both proteins belong to the SNF2 family of ATP-dependent DNA translocases important for a wide variety of chromatin remodeling activities. Despite the importance of this DNA motor activity in a number of DNA replication and repair activities, the mechanisms of translocation and how the motor is coupled to a specific remodeling function is unknown. Both SMARCAL1 and HLTF contain DNA- and protein- interacting domains immediately N-terminal to the conserved SNF2 motor. Our hypothesis is that specific interactions with DNA and other proteins (e.g., RPA) enforce selectivity for a particular type of stalled fork and drive fork regression through direct interactions with the junction. In Aim 1, we will investigate the general mechanisms of dsDNA translocation and fork reversal activities through structures and conformational states of the SMARCAL1 and HLTF motor apparatus in response to DNA binding and ATP turnover. In Aim 2, we will investigate how HLTF uses its HIRAN domain, a highly conserved yet uncharacterized DNA binding domain, in fork recognition and remodeling. In Aim 3, we will determine how specific interactions with DNA and RPA provide SMARCAL1 specificity for particular stalled fork structures. The structural and biochemical investigation proposed here will help determine the molecular mechanisms by which stalled replication forks are repaired, and will provide insight into a poorly understood family of DNA motor proteins critical for human health.