DNA strand annealing is promoted by the yeast Rad52 protein.
The Saccharomyces cerevisiae RAD52 gene plays a pivotal role in genetic recombination. Here we demonstrate that yeast Rad52 is a DNA binding protein. To show that the interaction between Rad52 and DNA is direct and not mediated by other yeast proteins and to facilitate protein purification, a recombinant expression system was developed. The recombinant protein can bind both single- and double-stranded DNA and the addition of either Mg2+ or ATP does not enhance the binding of single-stranded DNA. Furthermore, a DNA binding domain was found in the evolutionary conserved N terminus of the protein. More importantly, we show that the protein stimulates DNA annealing even in the presence of a large excess of nonhomologous DNA. Rad52-promoted annealing follows second-order kinetics and the rate is 3500-fold faster than that of the spontaneous reaction. How this annealing activity relates to the genetic phenotype associated with rad52 mutant cells is discussed.
Mortensen
U H
UH
Department of Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
Bendixen
C
C
Sunjevaric
I
I
Rothstein
R
R
D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000502:physiology*; D005656:Fungal Proteins / Q000502:physiology*; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D051721:Rad52 DNA Repair and Recombination Protein; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins