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Rar mutations which increase artificial chromosome stability in Saccharomyces cerevisiae identify transcription and recombination proteins.

ABSTRACT: In an attempt to identify trans-acting factors involved in replication origin function, we have characterized the RAR3 and RAR5 genes, identified by mutations which increase the mitotic stability of artificial chromosomes whose replication is dependent on the activity of weak ARS elements. Sequence analysis has shown that the RAR3 gene is identical to GAL11/SPT13, which encodes a putative transcription factor involved in the expression of a wide range of genes. Change-of-function mutations that truncate the RAR3 protein appear to be required to enhance chromosome stability. In contrast, loss of the RAR5 protein results in enhanced chromosome stability, as if the protein is an inhibitor of ARS function. The RAR5 gene encodes the 175 kDa DNA strand transfer protein beta, an activity that can promote the transfer of a strand from a double-stranded DNA molecule to a complementary single strand. This observation implies that a presumed recombination activity can affect eukaryotic chromosomal replication.

SUBMITTER: Kipling D

PROVIDER: S-EPMC333890 | biostudies-other | 1991 Apr

REPOSITORIES: biostudies-other

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rar mutations which increase artificial chromosome stability in Saccharomyces cerevisiae identify transcription and recombination proteins.

Kipling D D Tambini C C Kearsey S E SE

Nucleic acids research 19910401 7

In an attempt to identify trans-acting factors involved in replication origin function, we have characterized the RAR3 and RAR5 genes, identified by mutations which increase the mitotic stability of artificial chromosomes whose replication is dependent on the activity of weak ARS elements. Sequence analysis has shown that the RAR3 gene is identical to GAL11/SPT13, which encodes a putative transcription factor involved in the expression of a wide range of genes. Change-of-function mutations that ...[more]

PMID: 2027746

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Project description:BackgroundIn Saccharomyces cerevisiae, Rad59 is required for multiple homologous recombination mechanisms and viability in DNA replication-defective rad27 mutant cells. Recently, four rad59 missense alleles were found to have distinct effects on homologous recombination that are consistent with separation-of-function mutations. The rad59-K166A allele alters an amino acid in a conserved α-helical domain, and, like the rad59 null allele diminishes association of Rad52 with double-strand breaks. The rad59-K174A and rad59-F180A alleles alter amino acids in the same domain and have genetically similar effects on homologous recombination. The rad59-Y92A allele alters a conserved amino acid in a separate domain, has genetically distinct effects on homologous recombination, and does not diminish association of Rad52 with double-strand breaks.ResultsIn this study, rad59 mutant strains were crossed with a rad27 null mutant to examine the effects of the rad59 alleles on the link between viability, growth and the stimulation of homologous recombination in replication-defective cells. Like the rad59 null allele, rad59-K166A was synthetically lethal in combination with rad27. The rad59-K174A and rad59-F180A alleles were not synthetically lethal in combination with rad27, had effects on growth that coincided with decreased ectopic gene conversion, but did not affect mutation, unequal sister-chromatid recombination, or loss of heterozygosity. The rad59-Y92A allele was not synthetically lethal when combined with rad27, stimulated ectopic gene conversion and heteroallelic recombination independently from rad27, and was mutually epistatic with srs2. Unlike rad27, the stimulatory effect of rad59-Y92A on homologous recombination was not accompanied by effects on growth rate, cell cycle distribution, mutation, unequal sister-chromatid recombination, or loss of heterozygosity.ConclusionsThe synthetic lethality conferred by rad59 null and rad59-K166A alleles correlates with their inhibitory effect on association of Rad52 with double-strand breaks, suggesting that this may be essential for rescuing replication lesions in rad27 mutant cells. The rad59-K174A and rad59-F180A alleles may fractionally reduce this same function, which proportionally reduced repair of replication lesions by homologous recombination and growth rate. In contrast, rad59-Y92A stimulates homologous recombination, perhaps by affecting association of replication lesions with the Rad51 recombinase. This suggests that Rad59 influences the rescue of replication lesions by multiple recombination factors.

Dataset Information

Rar mutations which increase artificial chromosome stability in Saccharomyces cerevisiae identify transcription and recombination proteins.

Publications

rar mutations which increase artificial chromosome stability in Saccharomyces cerevisiae identify transcription and recombination proteins.

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