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Wednesday, July 29, 2020 | History

2 edition of Genetic and biochemical determinants of replication stress tolerance in Saccharomyces cerevisiae. found in the catalog.

Genetic and biochemical determinants of replication stress tolerance in Saccharomyces cerevisiae.

Robert Norman Woolstencroft

Genetic and biochemical determinants of replication stress tolerance in Saccharomyces cerevisiae.

by Robert Norman Woolstencroft

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Published .
Written in English


About the Edition

My combined genetic and biochemical strategy has forged novel determinants of RNR regulation and has enhanced our comprehension of the response to replication stress.The ribonucleotide reductase (RNR) inhibitor hydroxyurea (HU) depletes cellular pools of deoxyribonucleotides (dNTPs) and causes replication stress by pausing replication fork progression and blocking DNA synthesis. Stalled or collapsed DNA replication forks can lead to DNA double strand breaks, chromosome rearrangement, and loss of genome stability. The DNA replication checkpoint responds to replication stress by slowing S-phase progression, stabilizing stalled replication forks, and increasing RNR complex activity. In the budding yeast Saccharomyces cerevisiae, checkpoint responses hinge on activation of Mec1 (mammalian ATR ortholog) and Rad53 (mammalian Chk2 ortholog) checkpoint kinases.To identify novel gene activities that contribute to tolerance of replication stress, I surveyed the 4,812 strains in the S. cerevisiae non-essential haploid gene deletion collection for hypersensitivity to HU. Strains bearing deletions in either CCR4 or CAF1/POP2 , which encode components of the major cytoplasmic mRNA deadenylase complex, were amongst 49 gene deletions that confer susceptibility to replication stress. I found that Ccr4 cooperates with the Dun1 branch of the replication checkpoint, such that a ccr4Delta dun1Delta strain exhibits irreversible HU sensitivity and persistent Rad53 activation. Mutations in CRT1, which encodes the transcriptional repressor of RNR and DNA damage-induced genes, were uncovered as the major suppressors of ccr4Delta HU sensitivity. In addition, expression of RNR genes bypasses HU sensitivity of the ccr4Delta dun1Delta strain. These observations implicate coordinated regulation of Crt1 and RNR transcription via Ccr4 and Dun1 as a critical nodal point in the response to DNA replication stress.In parallel, I undertook a biochemical approach to interrogate the molecular function of Hug1, a small protein induced by DNA damage or replication stress. I found that Hug1 interacts with the small RNR proteins Rnr2 and Rnr4. Over-expression of HUG1 is lethal in combination with a dun1Delta mutation in the presence of HU; this lethal interaction requires a physical association of Hug1 with RNR subunits. I suggest a model whereby Hug1 is induced by HU and inhibits checkpoint responses via its physical interaction with RNR.

The Physical Object
Pagination280 leaves.
Number of Pages280
ID Numbers
Open LibraryOL21549464M
ISBN 109780494219386


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Genetic and biochemical determinants of replication stress tolerance in Saccharomyces cerevisiae by Robert Norman Woolstencroft Download PDF EPUB FB2