Human Rad52 facilitates a three-stranded pairing that follows no strand exchange: a novel pairing function of the protein.
Human Rad52 protein, by analogy with the genetics of yeast Rad52, is believed to mediate a pathway of homologous recombination even independent of Rad51. Current study is focused on unraveling the molecular properties of hRad52 that endow the protein such an ability. We show here that the hRad52 protein binds single-stranded DNA (ssDNA) as well as 3'- and 5'-tailed duplexes severalfold better than blunt-ended duplexes, altering the sensitivity of the bound DNA to the action of DNase I. Protein binding is sensitive to the length of the ssDNA: targets as short as a 33mer poorly bind the protein, whereas that of a 61mer and above bind the protein stably well. Such stable ssDNA-hRad52 complexes are highly competent in mediating not only the annealing of two complementary strands but also three-stranded pairing. The latter involves homologous recognition of linear duplex DNA by the ssDNA-hRad52 complex. We show that the hRad52 protein facilitates homologous recognition between ssDNA and duplex-DNA through a process that involves unwinding or transient unpairing of the interacting duplex via a novel three-stranded intermediate that does not lead to strand exchange. The results enable us to visualize a novel role for hRad52 that may model its function in a pathway requiring no hRad51.
Navadgi
Vasundhara M
VM
Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai- 400 005, India.
Dutta
Arnob
A
Rao
Basuthkar J
BJ
D020029:Base Pairing; D001667:Binding, Competitive; D003101:Collodion; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry; D029721:Drosophila Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D005374:Filtration / Q000295:instrumentation; D036341:Intercellular Signaling Peptides and Proteins; D009687:Nuclear Proteins / Q000378:metabolism; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D051721:Rad52 DNA Repair and Recombination Protein; D012689:Sequence Homology, Nucleic Acid; D013050:Spectrometry, Fluorescence
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C471550:Gcl protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D009692:Nucleic Acid Heteroduplexes; C497194:RAD52 protein, human; D051721:Rad52 DNA Repair and Recombination Protein; C076187:gcl protein, Drosophila; D003101:Collodion; D003850:Deoxyribonuclease I