Repair of a site-specifically placed psoralen crosslink, psoralen monoadduct, and double strand break in the yeast Saccharomyces cerevisiae.
Item
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Title
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Repair of a site-specifically placed psoralen crosslink, psoralen monoadduct, and double strand break in the yeast Saccharomyces cerevisiae.
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Identifier
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AAI9997092
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identifier
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9997092
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Creator
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Greenberg, Ross Bradley.
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Contributor
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Adviser: Wilma Saffran
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Date
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2001
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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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Chemistry, Biochemistry | Biology, Genetics
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Abstract
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Repair substrates carrying a single site-specifically placed psoralen crosslink, monoadduct, or double strand break were synthesized and used to compare repair, recombination, and gene conversion induced from each form of damage. The damage was located at the BsiWI site of a his3 allele carried on a plasmid. Damaged plasmids were introduced into yeast for in vivo repair. Genetic and physical analysis of colonies carrying repaired plasmids was performed.;The overall level of recombination and gene conversion induced from a DSB and a crosslink were similar. Monoadducts were non-recombinogenic. Similarities were observed for many of the properties of recombination and gene conversion induced by crosslinks and DSBs. These properties include the levels of reciprocal recombination, conversion tract length, the levels of uni- and bi-directional tracts, and the levels of discontinuous tracts. During crosslink and DSB induced gene conversion, the undamaged chromosomal allele was the primary donor of genetic information. The major difference in damage-induced recombination between crosslinks and DSBs was in the polarity of gene conversion tracts.;Gene conversion tracts induced from crosslinks exhibited a directional polarity, preferentially extending upstream from the damage site. Conversion tracts induced from a DSB also exhibited a polarity, but in the downstream rather than the upstream direction. The results show that the form of damage initiating the conversion influences the direction of the conversion tract and offer evidences that conversion tract polarity is linked to physical and/or mechanistic properties of the repair intermediate.;An additional difference between crosslinks and DSBs was in the occurrence of induced mutations. A psoralen crosslink, in addition to error-free repair, was a substrate for error-prone repair. Mutations at the damage site occurred in 9.1% of the non-reciprocal products. Mutations were not detected in the reciprocal products of crosslink repair. DSBs were non-mutagenic. These repair patterns indicate that there are two branches of crosslink repair. An error-free pathway is initiated by nucleotide excision repair, which processes crosslinks to DSBs. These DSBs are repaired by homologous recombination. An alternate, error-prone pathway also acts on crosslinks, and competes with the error-free branch for crosslink substrate. This error-prone branch generates targeted mutations.
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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.
