Functional Mapping of Integral and Peripheral Gene Involvement with the Proliferating Cell Nuclear Antigen-Dependent DNA Damage Tolerance Response in Saccharomyces cerevisiae

DNA damage is ubiquitous, arising from exogenous (genotoxic chemicals, free radical formation, radiation, and other factors) or endogenous (resulting from natural errors in cell cycle replication) influences. When damage occurs, essential cell processes are disrupted, resulting in aberrations in cell replication, the induction of carcinogenesis, the promotion of genetically based pathologies, or the progression into apoptotic or necrotic pathways. To preserve organism fitness, eukaryotes have developed multiple bypass or repair pathways to address genomic lesions or strand breaks. In this study, the budding yeast Saccharomyces (S.) cerevisiae is used as a model organism to investigate a highly conserved eukaryotic DNA damage tolerance (DDT) pathway involving the proliferating cell nuclear antigen (PCNA). Initial research in this project focused on investigating the critical components of this pathway relating to its three identified branches: (1) genomic preservation via sumoylation of PCNA, (2) initiation of translesion DNA synthesis (TLS) by mono-ubiquitination of PCNA, or (3) transition into error-free DDT by poly-ubiquitination of PCNA. By improving our understanding of the progression of these pathways and identifying any crosstalk between branches, I aimed to gain a more comprehensive understanding of this key cellular process. While researching this pathway, a novel interaction was also identified between yeast Sirtuin orthologs Sir3 and Sir4 with all branches of this DDT pathway. With human Sirtuins serving essential roles in maintaining cell homeostasis and replication, gene expression, and DNA damage responses, investigating the roles of yeast Sir3 and Sir4 in PCNA-dependent DDT could further clarify potential additional functions in cellular processes. Since yeast Sir proteins modulate the mating type expression of MATa or MATα at the homothallic mating type (HM) loci, the influence of mating type expression also needed to be explored. Budding yeast may switch from a natural haploid cell state to a diploid state expressing both mating types during environmental stress conditions to confer an advantage for cell survival. We postulated that a similar response may also occur in DNA damage responses, where mating type switching could contribute to the cell phenotype. As such, this research project aimed to (1) better define the PCNA dependent DDT response by investigating the key components for each identified branch in this pathway as well as identify any cross-talk that may be occurring, (2) clarify the novel function that Sir3 and Sir4 have independent of the Sir silencing complex with the DDT response, and (3) determine what role budding yeast mating type plays in damage responses. In vivo genetic knockouts were performed in S. cerevisiae strains via targeted genetic disruption with PCR-generated disruption cassettes. Gene manipulations in yeast cells were performed by transformation with plasmid vectors generated from Escherichia (E.) coli transformation and plasmid purification, which contained cloned genes, gene fragments, point-mutated genes, or yeast mating types. Transformed cells were selected using minimal media agar with drop-out medium corresponding to the integrated selectable marker gene and were then used for further experiments. Phenotype tests were conducted using serial dilution and gradient plate assays, as well as liquid-killing. Cells were exposed to multiple DNA-damaging agents, including methyl-methanesulfonate (MMS), 4-nitroquinoline oxide (4NQO), and ultraviolet (UV) radiation, to study alterations in cell survival. Further cell recombination efficiency (RE) was determined through integration of a pGAL:HO defective endonuclease plasmid. Lastly, gene expression was studied with reverse-transcriptase quantitative PCR (RT-qPCR). My results show that there is some crosstalk between the genomic integrity preservation branch via Srs2 and Siz1 with the DDT response branches TLS or error-free DDT via Rad5 and Rad18. Yeast Sir3 and Sir4, but not Sir2, were also shown to interact with all three identified branches. Our research determined that the novel independent function of Sir3 and Sir4 related to influencing the pathway switching between NHEJ and HR, where Sir3 and Sir4 appear to inherently repress HR and favour NHEJ progression. Lastly, yeast mating type appeared to influence cell survival, where heterozygous pseudo-diploid mutants were more resistant to DNA damage in sensitive DDT mutants. Though Sir3 and Sir4 also appeared to contribute directly to mating type expression both within and independent of the Sir silencing complex, the yeast mating type did not appear to further alter the tolerance response in conjunction with sirΔ null mutants. These findings clarify key eukaryotic damage response pathways by providing a deeper understanding of the progression of the PCNA-dependent DDT pathway. By identifying novel interactions of Sir3 and Sir4 peripheral to DDT, our understanding of yeast Sir proteins is expanded, providing insight into how they contribute to cellular DNA damage. We hope to apply these findings in a OneHealth context by extrapolating our findings to higher-level eukaryotes such as humans via linking findings in yeast Sir proteins and human orthologous Sirtuins.

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