Date

1-12-2009

UMMS Affiliation

Graduate School of Biomedical Sciences; Department of Biochemistry and Molecular Pharmacology

Document Type

Dissertation, Doctoral

Subjects

Methyl Methane sulfonate; DNA Damage; Cell Cycle Proteins; Intracellular Signaling Peptides and Proteins; Genes, cdc; S Phase; Academic Dissertations; Dissertations, UMMS

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

The main focus of my work has been the role of the MRN in the S-phase DNA damage checkpoint. The MRN plays many roles in cellular metabolism; some are checkpoint dependent and some are checkpoint independent. The multiple roles in cellular metabolism complicate study of the role of the MRN in the checkpoint. MRN mutations in budding yeast and mammals may display separation of function. Mechanistically, MRN, along with its cofactor Ctp1, is involved in 5’ resection to create single stranded DNA that is required for both signaling and homologous recombination. However, it is unclear if resection is essential for all of the cellular functions of MRN. Therefore I have made mutations to mimic those in budding yeast and mammals. I found that several alleles of rad32, as well as ctp1Δ, are defective in double-strand break repair and most other functions of the complex but maintain an intact S-phase DNA damage checkpoint. Thus, the MRN S-phase checkpoint role is separate from its Ctp1- and resection-dependent role in double-strand break repair. This observation leads me to conclude that other functions of MRN, possibly its role in replication fork metabolism, are required for S-phase DNA damage checkpoint function.

One of the potential roles of Rad32 and the rest of the MRN complex is in sister chromatid exchange. The genetic requirements of sister chromatid exchange have been examined using unequal sister chromatid assays which only are able to assay exchanges that are illegitimate and produce changes in the genome. Most sister chromatid exchange must be equal to maintain genomic integrity and thus far there is no good assay for equal sister chromatid exchange. Yeast cells expressing the human equilibrative nucleoside transporter 1 (hENT1) and the herpes simplex virus thymidine kinase (tk) are able to incorporate exogenous thymidine into their DNA. This strain makes it possible for the fission yeast DNA to be labeled with halogenated thymidine analogs. This strain is being used to design an assay that will label one sister with BrdU and then DNA combing will be used to see equal sister chromatid exchange.

 
 

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