Graduation Year

2023

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Kristina H. Schmidt, Ph.D.

Committee Member

Brant Burkhardt, Ph.D.

Committee Member

Marcus Cooke, Ph.D.

Committee Member

Younghoon Kee, Ph.D.

Committee Member

Patricia Kruk, Ph.D.

Keywords

Homologous Recombination, Base Excision Repair, Sod2, Genome Instability, DNA Repair

Abstract

Mutagenesis and genome instability are characteristics of cancer cells. People afflictedwith Bloom’s syndrome due to mutations in the BLM gene suffer from reduced life expectancy, accelerated aging, sensitivity to sun exposure, immune system abnormalities and increased risk of cancer. The predisposition to cancer is typically due to abnormal DNA repair that results in chromosome breakage and rearrangements. The homologous recombination factor Sgs1, which is a RecQ-like helicase and an ortholog of the human BLM helicase, functions primarily in DNA repair, but has also been implicated in other cellular functions like replication. Several functions of BLM such as its involvement in suppressing the oxidative stress phenotype have not been elucidated. Additionally, functional interactions of BLM with other DNA repair systems like base excision repair has not been studied. Here, we have used Saccharomyces cerevisiae as a model system and the BLM homolog SGS1 to understand the oxidative stress phenotype of cells lacking Sgs1 and the importance of Sgs1 in repairing DNA lesions other than double-strand breaks. The human MnSOD2 is a mitochondrial antioxidant enzyme that has been implicated in suppressing tumorigenesis. We show that its yeast homolog Sod2 suppresses mutagenesis and chromosomal rearrangements in the presence of exogenous oxidative stress. The mutagenesis is entirely dependent on the error-prone translesion DNA synthesis pathway whereas the chromosomal rearrangements are dependent on translesion DNA synthesis as well as homologous recombination factors like Rad51. Additionally, Sgs1 functionally interacts with Sod2 to suppress a subset of chromosomal rearrangements in the presence of exogenous oxidative stress, thereby preserving genome integrity. The genetic interactions of SOD2 with SGS1, RAD51, RAD59, REV3 and POL32 provide an overview of how lesions are repaired in cells lacking Sod2. Previous studies have alluded to the importance of homologous recombination in cells defective for base excision repair such as those lacking the AP endonucleases Apn1 and Apn2. However, the mechanism of repair or tolerance of lesions via homologous recombination has not been elucidated. Here, we have identified a novel role of Sgs1 in suppressing mutagenesis in the apn1Δ apn2Δ mutant. Through our genetic interaction studies of SGS1 and RAD51 with base excision repair genes APN1, APN2, NTG1, NTG2 and OGG1, we have established Sgs1 in two functionally separate pathways that deal with DNA lesions like abasic sites—the most spontaneously occurring lesions in the cell and highly mutagenic. Through a candidate screen we have also identified factors that suppress chromosomal rearrangements in cells lacking Apn1 and Apn2. Of all the DNA repair and metabolic factors tested, Pol32 was the most potent suppressor of chromosomal rearrangements in the apn1Δ apn2Δ mutant, suggesting that accumulation of DNA lesions and dysfunctional replication is a source of genome instability. Together, these studies identify novel cellular roles for Sgs1 by characterizing its interactions with antioxidant enzymes and the base excision repair pathway, both of which maintain genome integrity.

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