title: The SV40 large T-antigen origin binding domain directly participates in DNA unwinding.
abstract: The origin binding domain (OBD) of SV40 large T-ag serves a critical role during initiation of DNA replication to position T-ag on the origin. After origin recognition, T-ag forms a double hexamer over the origin. Within each hexamer, the OBD adopts a lock washer structure where the origin recognizing A1 and B2 loops face toward the helicase domain and likely become unavailable for binding DNA. In this study, we investigated the role of the central channel of the OBD hexamer in DNA replication by analyzing the effects of mutations of residues lining the channel. All mutants showed binding defects with origin DNA and ssDNA especially at low protein concentrations, but only half were defective at supporting DNA replication in vitro. All mutants were normal in unwinding linear origin DNA fragments. However, replication defective mutants failed to unwind a small origin containing circular DNA whereas replication competent mutants did so normally. The presence of RPA and/or pol/prim restored circular DNA unwinding activity of compromised mutants probably by interacting with the separated DNA strands on the T-ag surface. We interpret these results to indicate a role for the OBD central channel in binding and threading ssDNA during unwinding of circular SV40 DNA. Mixing experiments suggested that only one monomer in an OBD hexamer was necessary for DNA unwinding. We present a model of DNA threading through the T-ag complex illustrating how single-stranded DNA could pass close to a trough generated by key residues in one monomer of the OBD hexamer.
authors.affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware 19716-2590, USA.
authors.forename: Erin C
authors.initials: EC
authors.lastname: Foster
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Simmons
mesh_terms: D019943:Amino Acid Substitution; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004270:DNA, Circular / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013539:Simian virus 40
chemical_list: D000952:Antigens, Polyomavirus Transforming; D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004279:DNA, Viral