Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication.

Abstract

The simian virus 40 (SV40) large T antigen (large tumor antigen), in conjunction with a topoisomerase, a DNA binding protein, and ATP, catalyzed the conversion of a circular duplex DNA molecule containing the SV40 origin of replication to a form with unusual electrophoretic mobility that we have named form U. Analysis of this molecule revealed it-to be a highly underwound covalently closed circle. DNA unwinding was not detected with DNA containing a SV40 T-antigen binding site II mutation that renders the DNA inactive in replication. The unwinding reaction requires the action of a helicase, and SV40 T-antigen preparations contain such an activity. The T-antigen-associated ability to unwind DNA copurifiled with other activities intrinsic to T antigen [ability to support replication of SV40 DNA containing the SV40 origin, poly(dT)-stimulated ATPase activity, and DNA helicase]. However, in contrast to the unwinding activity, the SV40 T-antigen- This sequence of events is analogous to the steps proposed for the initiation of replication in prokaryotic systems. For instance, initiation of Xdv (8) and Escherichia coli origin (oriC) replication (9) is believed to depend upon precise protein interactions with the replication origin. These interactions enable the DnaB protein, a DNA helicase (10), to unwind the duplex. Furthermore, when a plasmid containing the E. coli oriC sequence was incubated with proteinsDnaA, DnaB, and DnaC, protein HU, single-strand DNA binding protein (SSB), and DNA gyrase-a DNA form of unusual electrophoretic mobility was produced (11). This form consisted of highly underwound covalently closed circles, and it was suggested that the helicase and DNA gyrase drive the unwinding.We have detected a similar origin-specific unwinding of DNA in a eukaryotic system, using SV40 origin-containing DNA. The protein requirements for the assay are T antigen, SSB, and a topoisomerase (topo) to relax the resultant supercoils. The reaction is also dependent on ATP, an ATP regenerating system, and Mg2". These observations suggested that T antigen is associated with a DNA helicase activity and that T antigen is responsible for the duplex DNA unwinding that is likely to initiate replication. While this work was in progress Stahl et al. (12) reported that SV40 T antigen contains a DNA helicase activity. MATERIALS AND METHODSAll reagents, enzyme preparations, and assay procedures used were as previously described (3) 16The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Homologous recombination enhancement conferred by the Z-DNA motif d(TG)30 is abrogated by simian virus 40 T antigen binding to adjacent DNA sequences.

“…After T-ag has bound specifically to binding sites I and II of the replication origin (8,9,46), it carries out an origin-dependent unwinding activity in the presence of ATP, MgCl2, and single-strand-binding protein (5,51). Furthermore the T-ag contains an intrinsic DNA helicase activity that can unwind duplex DNA distal to its binding sites (6,41,50). Thus, the origin localization, helix opening, and helicase activities are all provided solely by the T-ag, and, subsequently, other host factors are required to complete the replication process (reviewed in reference 18).…”

Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding

“…Crystal structures of Orc1 complexed with dsDNA suggest that a dimer binds dsDNA through the winged-helix domain of the protein in an ATP-dependent fashion; this complex distorts DNA base pairing, which facilitates the recruitment and loading of the MCM complex (57). Unlike DnaB, which requires accessory factors, the simian virus 40 (SV40) and T4 phage Dda helicase can initiate DNA unwinding from an origin sequence (59)(60)(61)(62). Specifically, SV40 has been shown to initiate unwinding and proceed bidirectionally to unwind plasmid length DNA (59).…”

In the Simian Virus 40 In Vitro Replication System, Start Site Selection by the Polymerase α-Primase Complex Is Not Significantly Altered by Changes in the Concentration of Ribonucleotides

“…SV40 replication is initiated when T antigen (T-ag), the single viral protein necessary for replication, site specifically binds to the viral origin (reviewed in references 8, 11, and 31). Upon binding, T-ag assembles into a double hexamer (21,23,51,61,77) that is able to function as a helicase (22,34,66,68,84). Owing to its helicase activity, T-ag is capable of catalyzing origin-specific unwinding, provided replication protein A (RPA) and topoisomerase I are present in the reaction (15,22,27,88).…”