Denaturation of the simian virus 40 origin of replication mediated by human replication protein A.
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.
Iftode
C
C
Department of Biochemistry and Kaplan Comprehensive Cancer Center, New York University Medical Center, New York 10016, USA.
Borowiec
J A
JA
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
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases