Date

September 2009

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Molecular and Medical Genetics

Institution

Oregon Health & Science University

Abstract

Although it is well established that DNA-protein crosslinks are formed as a consequence of cellular exposure to agents such as formaldehyde, transplatin, ionizing and ultraviolet radiation, the biochemical pathways that promote cellular survival via repair or tolerance of these lesions are poorly understood. To investigate the mechanisms that function to limit DNA-protein crosslink-induced cytotoxicity, the Saccharomyces cerevisiae non-essential gene deletion library was screened for increased sensitivity to formaldehyde exposure. Following low-dose, chronic exposure, strains containing deletions in genes mediating homologous recombination showed the greatest sensitivity, while under the same exposure conditions, deletions in genes associated with nucleotide excision repair conferred only low to moderate sensitivities. However, when the exposure regime was changed to a high-dose acute (short-term) formaldehyde treatment, the genes that conferred maximal survival switched to the nucleotide excision repair pathway, with little contribution of the homologous recombination genes. Data are presented which suggest that following acute formaldehyde exposure, repair and/or tolerance of DNA-protein crosslinks proceeds via formation of nucleotide excision repair-dependent single strand break intermediates and without a detectable accumulation of double strand breaks. In addition, the relative survivals of the formaldehyde sensitive deletion strains were assessed following exposure to other DPC-inducing agents. Not only do the exposure conditions influence the cellular response, but also the specific agent used to induce the damage. Based on the results of the genome-wide screen, the interactions of the implicated pathways were investigated. Genetic analyses were performed by creating yeast strains with deletions in genes from both the classically defined nucleotide excisions repair and homologous recombination pathways. Based on these results, it appears that in the absence of NER and HR, the translesion synthesis pathway may function as a backup tolerance pathway to enhance cell survival after formaldehyde exposure. In addition, both Mre11 and Rad1, independent from the other homologous recombination and nucleotide excision repair proteins, play a separate role in the tolerance and/or repair of DNA-protein crosslinks. These data clearly demonstrate a differential pathway response to chronic versus acute formaldehyde exposures and may have significance and implications for risk extrapolation in human exposure studies.

Identifier

doi:10.6083/M41N7Z38

School

School of Medicine

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