Recombinational repair of double-strand breaks in the yeast Saccharomyces cerevisiae. The role of RAD51, RAD54, RAD55, and RAD57.
Item
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Title
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Recombinational repair of double-strand breaks in the yeast Saccharomyces cerevisiae. The role of RAD51, RAD54, RAD55, and RAD57.
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Identifier
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AAI9946215
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identifier
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9946215
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Creator
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Roccanova, Louis.
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Contributor
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Adviser: Wilma Saffran
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Date
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1999
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular | Biology, Genetics | Health Sciences, Oncology
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Abstract
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Double-strand breaks in chromosomal DNA are lethal to cells. They are repaired primarily by homologous recombination in the yeast Saccharomyces cerevisiae. The double-strand break repair model has been proposed as a mechanism for this repair (Szostak et. al. 1983 Cell 33, 26-36). Many of the proteins involved are proposed to make up a multimer referred to as the 'recombinosome'. Strains mutated in genes of the RAD52 epistasis group have been shown to be sensitive to double-strand damage. Due to various interactions with each other, their products are proposed to be part of the recombinosome.;This is a study of double-strand break repair of plasmids in the yeast Saccharomyces cerevisiae. We have analyzed double-strand break induced gene conversion and crossover recombination between plasmids and chromosomes. Experiments were conducted in wild type and recombinational repair deficient strains, rad5l, rad54, rad55, and rad57.;Plasmids were constructed which carry the TRP1 gene, and one of five nonfunctional alleles of the HIS3 gene. These his3 genes contain frameshift mutations made by the insertion of a restriction site marker at different locations. The plasmids were linearized and transformed into wild type and recombinational repair deficient strains. Colonies containing repaired plasmids were examined for recombination by selective plating and PCR analysis. Double strand breaks were found to induce both gene conversion and plasmid integration. The gene products of RAD51 and RAD54 were shown to be essential in recombinational repair while RAD55 and RAD57 were shown to play an auxiliary role. In the wild type strain, conversion occurred in both directions from uncut to linearized DNA and from linearized to uncut DNA. Conversion tract lengths were at least 13 base pairs, and as many as 457 base pairs. In repair events in recombinational repair deficient strains, we observed complete marker recovery in the absence of gene conversion. This suggests the existence of a second repair pathway for the repair of double strand breaks. The patterns of gene conversion leading to His+ colonies were consistant with the predictions of the double-strand break repair model.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
