title: Denaturation of the simian virus 40 origin of replication mediated by human replication protein A.
abstract: The initiation of simian virus 40 (SV40) replication requires recognition of the viral origin of replication (ori) by SV40 T antigen, followed by denaturation of ori in a reaction dependent upon human replication protein A (hRPA). To understand how origin denaturation is achieved, we constructed a 48-bp SV40 "pseudo-origin" with a central 8-nucleotide (nt) bubble flanked by viral sequences, mimicking a DNA structure found within the SV40 T antigen-ori complex. hRPA bound the pseudo-origin with similar stoichiometry and an approximately fivefold reduced affinity compared to the binding of a 48-nt single-stranded DNA molecule. The presence of hRPA not only distorted the duplex DNA flanking the bubble but also resulted in denaturation of the pseudo-origin substrate in an ATP-independent reaction. Pseudo-origin denaturation occurred in 7 mM MgCl2, distinguishing this reaction from Mg2+-independent DNA-unwinding activities previously reported for hRPA. Tests of other single-stranded DNA-binding proteins (SSBs) revealed that pseudo-origin binding correlates with the known ability of these SSBs to support the T-antigen-dependent origin unwinding activity. Our results suggest that hRPA binding to the T antigen-ori complex induces the denaturation of ori including T-antigen recognition sequences, thus releasing T antigen from ori to unwind the viral DNA. The denaturation activity of hRPA has the potential to play a significant role in other aspects of DNA metabolism, including DNA repair.
authors.affiliation: Department of Biochemistry and Kaplan Comprehensive Cancer Center, New York University Medical Center, New York 10016, USA.
authors.forename: C
authors.initials: C
authors.lastname: Iftode
authors.forename: J A
authors.initials: JA
authors.lastname: Borowiec
mesh_terms: D001483:Base Sequence; D004265:DNA Helicases / Q000378:metabolism; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000648:ultrastructure; D004279:DNA, Viral / Q000648:ultrastructure*; D004268:DNA-Binding Proteins / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D006801:Humans; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D011506:Proteins / Q000502:physiology*; D018741:Replication Origin; D013539:Simian virus 40 / Q000235:genetics*; D015534:Trans-Activators
chemical_list: D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases