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David Alvaro(2), Michael Lisby(1), Rodney
Rothstein(1). (1) Genetics and Development, Columbia University, 701
W. 168th St., New York, NY, 10032, USA; (2) 701 W 168th St, HHSC
1606, New York, NY 10032.
Relocalization of a Rad52-YFP fusion protein to subnuclear foci can
be used as a marker for recombinational DNA repair. In mitotically
growing cells, spontaneous foci form at a low frequency. These foci
likely reflect the formation of repair centers in response to
spontaneous DNA lesions. Mutations in various pathways of DNA
metabolism lead to elevations in the frequency of Rad52 foci. To
detect additional genes affecting DNA repair processes, we began
screening the disruption library by introducing a Rad52-YFP reporter
and assaying focus formation. Initial results revealed that
recessive genetic factors in the library strains, independent of the
documented gene disruptions, influence the Rad52 focus phenotype. To
minimize background effects, a method was developed to eliminate
recessive factors in the library background. This is accomplished by
creating hybrid diploids homozygous for the gene deletion by mating
each library strain to a W303 strain containing a conditional wild
type copy of the chromosome bearing the gene disruption, which
itself can be selectively lost after mating. Over 2400 diploid
strains have been screened for Rad52-YFP focus levels and 44 gene
deletions have been identified that exhibit significantly elevated
focus levels. The genes identified in the screen reflect many
pathways of DNA metabolism including replication, repair,
recombination, sister chromatid cohesion, chromosome segregation,
and transcription, as well as several uncharacterized genes.
Program Nr. 492C from 2004 Yeast meeting |
