Date of Award

8-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Committee Chair/Advisor

Michael Sehorn

Committee Member

Jennifer Mason

Committee Member

Kimberly Paul

Committee Member

Kerry Smith

Abstract

The genome is constantly at risk to damage from environmental and normal cellular processes. Double-strand breaks (DSBs) are one of the most deleterious forms of DNA damage as unrepaired DSBs ultimately result in cell death. Homologous recombination (HR) is a conserved DNA repair pathway that utilizes a homologous chromosome as a template for DNA synthesis to repair the DSBs relatively error-free. HR is active in both mitotic and meiotic cells but meiotic HR is initiated by the introduction of programmed DSBs into the genome. The process of HR repairs the DSBs and results in a physical connection between homologous chromosomes called synapsis. Proper segregation of chromosomes is essential to ensure each daughter cell has the complete set of chromosomes. Defective meiotic HR can lead to an abnormal distribution of chromosomes in each daughter cell which may result in genetic disorders and infertile sex cells. Much of what is known about the meiotic HR pathway is based on studies in yeast. Some protein factors in meiotic HR are conserved between yeast and mammals, while others are unique to mammalian meiotic HR. Yeast and humans both recruit a single-strand DNA (ssDNA) binding protein, replication protein A (RPA), to DSBs upon recognition of damage. In higher eukaryotes, two additional ssDNA binding proteins called hSSB1 and hSSB2 localize to the site of DSBs. hSSB1 is present in both mitotic and meiotic cells whereas hSSB2 is primarily expressed in meiotic cells. While hSSB1 has a role in DNA damage recognition in mitotic cells, little is known about the role hSSB1 or hSSB2 have in meiosis. Both hSSB1 and hSSB2 and RPA bind to the 3’ ssDNA ends exposed after the enzymatic processing of a DSB to protect the ends from forming secondary structure and to prevent digestion by nucleases. The tumor suppressor protein BRCA2 displaces RPA from the resected 3’ ssDNA end to load the RAD51 and DMC1 recombinases. MEILB2 is a meiosis-specific protein that localizes BRCA2 to the resected 3’ ssDNA ends, but the mechanism by which MEILB2 functions with BRCA2 is poorly understood. The recombinase-bound nucleoprotein filaments search the chromatin loops of the homologous chromosome for complementary sequences to use as a repair template. These chromatin loops are bridged together by structural proteins such as SYCP3 which also interact with the RAD51 and DMC1 recombinases. The significance of this SYCP3-recombinase interaction is not well understood. Biochemical characterization of SSB1, SSB2, SYCP3 and MEILB2 will shed light on the mechanisms by which these proteins function in meiotic recombination.

Author ORCID Identifier

0000-0003-4292-874X

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