Studies of the interaction between Rad52 protein and the yeast single-stranded DNA binding protein RPA.
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. A Saccharomyces cerevisiae strain carrying the rfa1-44 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, placing RFA1 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.
Hays
S L
SL
Department of Biochemistry, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford University, Stanford, California 94305, USA.
Firmenich
A A
AA
Massey
P
P
Banerjee
R
R
Berg
P
P
D000483:Alleles; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004843:Epistasis, Genetic; D005656:Fungal Proteins / Q000378:metabolism*; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D051135:Rad51 Recombinase; D051721:Rad52 DNA Repair and Recombination Protein; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C497197:RAD52 protein, S cerevisiae; C082727:RFA2 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D016695:Glycosyltransferases; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase