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.

Protein-Protein Interactions between the Escherichia coli Single-Stranded DNA-Binding Protein and Exonuclease I

1996 ◽  
Vol 145 (5) ◽  
pp. 619 ◽  
Author(s):  
Margarita Sandigursky ◽  
Frances Mendez ◽  
Robert E. Bases ◽  
Tomohiro Matsumoto ◽  
William A. Franklin
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Protein Interactions ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Protein Protein Interactions ◽  
Single Stranded Dna ◽  
Exonuclease I

scholarly journals FANCJ (BACH1) helicase forms DNA damage inducible foci with replication protein A and interacts physically and functionally with the single-stranded DNA-binding protein

Blood ◽  
2007 ◽  
Vol 110 (7) ◽  
pp. 2390-2398 ◽  
Author(s):  
Rigu Gupta ◽  
Sudha Sharma ◽  
Joshua A. Sommers ◽  
Mark K. Kenny ◽  
Sharon B. Cantor ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Binding Protein ◽  
Critical Role ◽  
Protein A ◽  
Replication Protein A ◽  
Dna Binding Protein ◽  
Replication Protein ◽  
Single Stranded Dna

The BRCA1 associated C-terminal helicase (BACH1, designated FANCJ) is implicated in the chromosomal instability genetic disorder Fanconi anemia (FA) and hereditary breast cancer. A critical role of FANCJ helicase may be to restart replication as a component of downstream events that occur during the repair of DNA cross-links or double-strand breaks. We investigated the potential interaction of FANCJ with replication protein A (RPA), a single-stranded DNA-binding protein implicated in both DNA replication and repair. FANCJ and RPA were shown to coimmunoprecipitate most likely through a direct interaction of FANCJ and the RPA70 subunit. Moreover, dependent on the presence of BRCA1, FANCJ colocalizes with RPA in nuclear foci after DNA damage. Our data are consistent with a model in which FANCJ associates with RPA in a DNA damage-inducible manner and through the protein interaction RPA stimulates FANCJ helicase to better unwind duplex DNA substrates. These findings identify RPA as the first regulatory partner of FANCJ. The FANCJ-RPA interaction is likely to be important for the role of the helicase to more efficiently unwind DNA repair intermediates to maintain genomic stability.

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 Mutagenesis of the Agrobacterium VirE2 Single-Stranded DNA-Binding Protein Identifies Regions Required for Self-Association and Interaction with VirE1 and a Permissive Site for Hybrid Protein Construction

1999 ◽  
Vol 181 (14) ◽  
pp. 4342-4352 ◽  
Author(s):  
Xue-Rong Zhou ◽  
Peter J. Christie
Keyword(s):  
Dna Binding ◽  
Protein Function ◽  
Tertiary Structure ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Single Stranded Dna ◽  
Mutant Proteins ◽  
C Terminus ◽  
Self Association ◽  
Peptide Insertion

ABSTRACT The VirE2 single-stranded DNA-binding protein (SSB) ofAgrobacterium tumefaciens is required for delivery of T-DNA to the nuclei of susceptible plant cells. By yeast two-hybrid and immunoprecipitation analyses, VirE2 was shown to self-associate and to interact with VirE1. VirE2 mutants with small deletions or insertions of a 31-residue oligopeptide (i31) at the N or C terminus or with an i31 peptide insertion at Leu236 retained the capacity to form homomultimers. By contrast, VirE2 mutants with modifications outside a central region located between residues 320 and 390 retained the capacity to interact with VirE1. These findings suggest the tertiary structure of VirE2 is important for homomultimer formation whereas a central domain mediates formation of a complex with VirE1. The capacity of VirE2 mutants to interact with full-length VirE2 in the yeastSaccharomyces cerevisiae correlated with the abundance of the mutant proteins in A. tumefaciens, suggesting that VirE2 is stabilized by homomultimerization in the bacterium. We further characterized the promoter and N- and C-terminal sequence requirements for synthesis of functional VirE2. APvirB ::virE2 construct yielded functional VirE2 protein as defined by complementation of avirE2 null mutation. By contrast,PvirE or Plac promoter constructs yielded functional VirE2 only if virE1 was coexpressed with virE2. Deletion of 10 or 9 residues from the N or C terminus of VirE2, respectively, or addition of heterologous peptides or proteins to either terminus resulted in a loss of protein function. However, an i31 peptide insertion at Tyr39 had no effect on protein function as defined by the capacity of the mutant protein to (i) interact with native VirE2, (ii) interact with VirE1, (iii) accumulate at abundant levels in A. tumefaciens, and (iv) restore wild-type virulence to a virE2 null mutant. We propose that Tyr39 of VirE2 corresponds to a permissive site for insertion of heterologous peptides or proteins of interest for delivery across kingdom boundaries.

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