Date of Award

8-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Committee Chair/Advisor

Jennifer Mason

Committee Member

Michael Sehorn

Committee Member

Meredith Morris

Committee Member

Andrei Alexandra

Abstract

The genome must be accurately and efficiently replicated each cell cycle to ensure there is not accumulation of mutations or structural rearrangements. When an active replication fork stalls, there are multiple DNA damage tolerance (DTT) pathways that can act to both protect integrity of DNA at the fork and restart replication. RAD51, a vital recombinase, works with HLTF, ZRANB3, and SMARCAL1 or FBH1 to regress stalled replication forks. RAD51 is loaded and stabilized onto the replication fork to protect the newly synthesized DNA from degradation. RAD51 is also active during the template switching DDT pathway. This pathway can be utilized to restart stalled forks independently of a DNA break or during break-induced replication. Despite the fact that RAD51 plays such a vital role during the replication stress response of cells, how it is regulated at replication forks has not been as well-characterized. Here, we studied various mechanisms of the regulation of RAD51 during replication stress. We determined that RAD51-dependent template switching promotes replication stress resistance in FBH1-deficient cells. Then, we narrowed the RAD51 interaction domain on FBH1 to 25 amino acids on the N-terminus. Finally, we provided evidence that BRCA2 and FANCD2 load and stabilize RAD51 onto structurally distinct replication intermediates following fork regression. This work has provided mechanistic insight into how RAD51 is controlled at stalled replication forks.

Author ORCID Identifier

0000-0003-0480-7082

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