scholarly journals A mutation in the gene encoding the Saccharomyces cerevisiae single-stranded DNA-binding protein Rfa1 stimulates a RAD52-independent pathway for direct-repeat recombination.

1995 ◽  
Vol 15 (3) ◽  
pp. 1632-1641 ◽  
Author(s):  
J Smith ◽  
R Rothstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Binding Protein ◽  
Large Subunit ◽  
Direct Repeat ◽  
Fold Increase ◽  
Dna Binding Protein ◽  
Yeast Saccharomyces Cerevisiae ◽  
Single Stranded Dna ◽  
Mutant Strains

In the yeast Saccharomyces cerevisiae, recombination between direct repeats is synergistically reduced in rad1 rad52 double mutants, suggesting that the two genes define alternate recombination pathways. Using a classical genetic approach, we searched for suppressors of the recombination defect in the double mutant. One mutation that restores wild-type levels of recombination was isolated. Cloning by complementation and subsequent physical and genetic analysis revealed that it maps to RAF1. This locus encodes the large subunit of the single-stranded DNA-binding protein complex, RP-A, which is conserved from S. cerevisiae to humans. The rfa1 mutation on its own causes a 15-fold increase in direct-repeat recombination. However, unlike most other hyperrecombination mutations, the elevated levels in rfa1 mutants occur independently of RAD52 function. Additionally, rfa1 mutant strains grow slowly, are UV sensitive, and exhibit decreased levels of heteroallelic recombination. DNA sequence analysis of rfa1 revealed a missense mutation that alters a conserved residue of the protein (aspartic acid 228 to tyrosine [D228Y]). Biochemical analysis suggests that this defect results in decreased levels of RP-A in mutant strains. Overexpression of the mutant subunit completely suppresses the UV sensitivity and partially suppresses the recombination phenotype. We propose that the defective complex fails to interact properly with components of the repair, replication, and recombination machinery. Further, this may permit the bypass of the recombination defect of rad1 rad52 mutants by activating an alternative single-stranded DNA degradation pathway.

scholarly journals An essential Saccharomyces cerevisiae single-stranded DNA binding protein is homologous to the large subunit of human RP-A.

1990 ◽  
Vol 9 (7) ◽  
pp. 2321-2329 ◽  
Author(s):  
W. D. Heyer ◽  
M. R. Rao ◽  
L. F. Erdile ◽  
T. J. Kelly ◽  
R. D. Kolodner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Binding Protein ◽  
Large Subunit ◽  
Dna Binding Protein ◽  
Single Stranded Dna

scholarly journals Synergistic release from glucose repression by mig1 and ssn mutations in Saccharomyces cerevisiae.

Genetics ◽  
1994 ◽  
Vol 137 (1) ◽  
pp. 49-54 ◽  
Author(s):  
L G Vallier ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Dna Binding ◽  
Glucose Repression ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Yeast Saccharomyces Cerevisiae ◽  
Suc2 Promoter ◽  
Repressor Complex ◽  
Glucose Availability

Abstract In the yeast Saccharomyces cerevisiae, glucose repression of SUC2 transcription requires the SSN6-TUP1 repressor complex. It has been proposed that the DNA-binding protein MIG1 secures SSN6-TUP1 to the SUC2 promoter. Here we show that a mig1 deletion does not cause nearly as dramatic a loss of repression as ssn6: glucose-grown mig1 mutants display 20-fold lower SUC2 expression than ssn6 mutants. Thus, repression by SSN6-TUP1 is not mediated solely by MIG1, but also involves MIG1-independent mechanisms. We report that mig1 partially restores SUC2 expression in mutants lacking the SNF1 protein kinase and show that mig1 is allelic to ssn1, a mutation selected as a suppressor of snf1. Other SSN genes identified in this selection were therefore candidates for a role in repression of SUC2. We show that mig1 acts synergistically with ssn2 through ssn5, ssn7, and ssn8 to relieve glucose repression of SUC2 and to suppress the requirement for SNF1. These findings indicate that the SSN proteins contribute to repression of SUC2, and the pleiotropic phenotypes of the ssn mutants suggest global roles in repression. Finally, the regulated SUC2 expression observed in snf1 mig1 mutants indicates that signals regarding glucose availability can be transmitted independently of the SNF1 protein kinase.

scholarly journals Identification of a Saccharomyces cerevisiae DNA-binding protein involved in transcriptional regulation.

1990 ◽  
Vol 10 (4) ◽  
pp. 1743-1753 ◽  
Author(s):  
H Wang ◽  
P R Nicholson ◽  
D J Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Rrna Gene ◽  
Yeast Saccharomyces Cerevisiae ◽  
Yeast Genes

A DNA-binding protein has been identified from extracts of the budding yeast Saccharomyces cerevisiae which binds to sites present in the promoter regions of a number of yeast genes transcribed by RNA polymerase II, including SIN3 (also known as SDI1), SWI5, CDC9, and TOP1. This protein also binds to a site present in the enhancer for the 35S rRNA gene, which is transcribed by RNA polymerase I, and appears to be identical to the previously described REB1 protein (B. E. Morrow, S. P. Johnson, and J. R. Warner, J. Biol. Chem. 264:9061-9068, 1989). When oligonucleotides containing a REB1-binding site are placed between the CYC1 upstream activating sequence and TATA box, transcription by RNA polymerase II in vivo is substantially reduced, suggesting that REB1 acts as a repressor of RNA polymerase II transcription. The in vitro levels of the REB1 DNA-binding activity are reduced in extracts prepared from strains bearing a mutation in the SIN3 gene. A greater reduction in REB1 activity is observed if the sin3 mutant strain is grown in media containing galactose as a carbon source.

Identification of a Saccharomyces cerevisiae DNA-binding protein involved in transcriptional regulation

1990 ◽  
Vol 10 (4) ◽  
pp. 1743-1753
Author(s):  
H Wang ◽  
P R Nicholson ◽  
D J Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Rrna Gene ◽  
Yeast Saccharomyces Cerevisiae ◽  
Yeast Genes

A DNA-binding protein has been identified from extracts of the budding yeast Saccharomyces cerevisiae which binds to sites present in the promoter regions of a number of yeast genes transcribed by RNA polymerase II, including SIN3 (also known as SDI1), SWI5, CDC9, and TOP1. This protein also binds to a site present in the enhancer for the 35S rRNA gene, which is transcribed by RNA polymerase I, and appears to be identical to the previously described REB1 protein (B. E. Morrow, S. P. Johnson, and J. R. Warner, J. Biol. Chem. 264:9061-9068, 1989). When oligonucleotides containing a REB1-binding site are placed between the CYC1 upstream activating sequence and TATA box, transcription by RNA polymerase II in vivo is substantially reduced, suggesting that REB1 acts as a repressor of RNA polymerase II transcription. The in vitro levels of the REB1 DNA-binding activity are reduced in extracts prepared from strains bearing a mutation in the SIN3 gene. A greater reduction in REB1 activity is observed if the sin3 mutant strain is grown in media containing galactose as a carbon source.

scholarly journals Studies of the Interaction between Rad52 Protein and the Yeast Single-Stranded DNA Binding Protein RPA

1998 ◽  
Vol 18 (7) ◽  
pp. 4400-4406 ◽  
Author(s):  
Sharon L. Hays ◽  
Antoine A. Firmenich ◽  
Philip Massey ◽  
Ronadip Banerjee ◽  
Paul Berg
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Binding Protein ◽  
Large Subunit ◽  
Critical Role ◽  
Dna Binding Protein ◽  
Protein Protein Interactions ◽  
Single Stranded Dna ◽  
Mutant Proteins ◽  
Two Hybrid

ABSTRACT The RFA1 gene encodes the large subunit of the yeast trimeric single-stranded DNA binding protein replication protein A (RPA), which is known to play a critical role in DNA replication. ASaccharomyces cerevisiae strain carrying therfa1-44 allele displays a number of impaired recombination and repair phenotypes, all of which are suppressible by overexpression of RAD52. We demonstrate that a rad52 mutation is epistatic to the rfa1-44 mutation, placingRFA1 and RAD52 in the same genetic pathway. Furthermore, two-hybrid analysis indicates the existence of interactions between Rad52 and all three subunits of RPA. The nature of this Rad52-RPA interaction was further explored by using two different mutant alleles of rad52. Both mutations lie in the amino terminus of Rad52, a region previously defined as being responsible for its DNA binding ability (U. H. Mortenson, C. Beudixen, I. Sunjeuaric, and R. Rothstein, Proc. Natl. Acad. Sci. USA 93:10729–10734, 1996). The yeast two-hybrid system was used to monitor the protein-protein interactions of the mutant Rad52 proteins. Both of the mutant proteins are capable of self-interaction but are unable to interact with Rad51. The mutant proteins also lack the ability to interact with the large subunit of RPA, Rfa1. Interestingly, they retain their ability to interact with the medium-sized subunit, Rfa2. Given the location of the mutations in the DNA binding domain of Rad52, a model incorporating the role of DNA in the protein-protein interactions involved in the repair of DNA double-strand breaks is presented.

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