title: The single-stranded DNA binding protein of Sulfolobus solfataricus acts in the presynaptic step of homologous recombination.
abstract: Homologous recombination is an important pathway in the repair of DNA double-strand breaks in all organisms. In mesophiles, single-stranded DNA binding proteins (SSBs) are believed to be involved in the removal of single-stranded DNA (ssDNA) secondary structure during the presynaptic step of homologous recombination, facilitating the formation of a contiguous Rad51/RecA nucleoprotein filament. Here we report a role for the thermophilic archaeal Sulfolobus solfataricus SSB (SsoSSB) in the presynaptic step of homologous recombination. We have identified multiple quaternary structural forms of this protein in vivo and examined the activity of SsoSSB with the strand-exchange protein S. solfataricus RadA (SsoRadA). Using gel-shift analysis, we found that the two major forms of SsoSSB have different DNA binding affinities and site sizes. Biochemical examination of the monomeric form of SsoSSB suggests that it has a minor role in presynapsis and may slightly inhibit the ssDNA-dependent ATPase activity of SsoRadA. The tetrameric form of SsoSSB, however, significantly inhibits SsoRadA ssDNA-dependent ATPase activity under both saturating and subsaturating conditions. Order-of-addition experiments indicate that preincubation of tetrameric SsoSSB and SsoRadA prior to reaction initiation with ssDNA relieves the inhibition observed when SsoSSB is added either before or after SsoRadA. In addition, we demonstrate a direct interaction between SsoRadA and SsoSSB using coimmunoprecipitation. Taken together, these results suggest that a direct interaction between SsoSSB and SsoRadA may occur in vivo prior to the formation of the SsoRadA nucleoprotein filament.
authors.affiliation: School of Molecular Biosciences, Washington State University, Pullman, WA 99163, USA.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Rolfsmeier
authors.forename: Cynthia A
authors.initials: CA
authors.lastname: Haseltine
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000906:Antibodies / Q000494:pharmacology; D019843:Archaeal Proteins / Q000037:antagonists & inhibitors; D002850:Chromatography, Gel; D023902:Chromosome Pairing / Q000187:drug effects; D003429:Cross Reactions / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005453:Fluorescence; D047468:Immunoprecipitation; D008954:Models, Biological; D011485:Protein Binding / Q000187:drug effects; D020033:Protein Isoforms / Q000378:metabolism; D055503:Protein Multimerization / Q000187:drug effects; D011995:Recombination, Genetic / Q000187:drug effects; D048229:Sulfolobus solfataricus / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D013696:Temperature
chemical_list: D000906:Antibodies; D019843:Archaeal Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D020033:Protein Isoforms; C117252:RadA protein, archaeal; D000251:Adenosine Triphosphatases; C037693:single stranded DNA dependent ATPase