The DNA binding preference of RAD52 and RAD59 proteins: implications for RAD52 and RAD59 protein function in homologous recombination.
We examined the double-stranded DNA (dsDNA) binding preference of the Saccharomyces cerevisiae Rad52 protein and its homologue, the Rad59 protein. In nuclease protection assays both proteins protected an internal sequence and the dsDNA ends equally well. Similarly, using electrophoretic mobility shift assays, we found the affinity of both Rad52 and Rad59 proteins for DNA ends to be comparable with their affinity for internal sequences. The protein-DNA complexes were also directly visualized using atomic force microscopy. Both proteins formed discrete complexes, which were primarily found (90-94%) at internal dsDNA sites. We also measured the DNA end binding behavior of human Rad52 protein and found a slight preference for dsDNA ends. Thus, these proteins have no strong preference for dsDNA ends over internal sites, which is inconsistent with their function at a step of dsDNA break repair that precedes DNA processing. Therefore, we conclude that S. cerevisiae Rad52 and Rad59 proteins and their eukaryotic counterparts function by binding to single-stranded DNA formed as intermediates of recombination rather than by binding to the unprocessed DNA double-strand break.
Wu
Yun
Y
Section of Microbiology, Center for Genetics and Development, University of California, Davis, California 95616-8665, USA.
Siino
Joseph S
JS
Sugiyama
Tomohiko
T
Kowalczykowski
Stephen C
SC
D053903:DNA Breaks, Double-Stranded; D004271:DNA, Fungal / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D011485:Protein Binding / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000235:genetics; D051721:Rad52 DNA Repair and Recombination Protein / Q000378:metabolism* / Q000502:physiology; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism* / Q000502:physiology; D012689:Sequence Homology, Nucleic Acid
D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C497197:RAD52 protein, S cerevisiae; C497194:RAD52 protein, human; C400213:RAD59 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins