ATP stimulates the binding of simian virus 40 (SV40) large tumor antigen to the SV40 origin of replication.

Abstract

Simian virus 40 (SV40) large tumor antigen (T antigen) Past studies have indicated that there are two major binding sites for T antigen located in the ori region, designated binding sites I and II (9). T antigen has a 20-to 30-fold higher affinity for site I than site 11 (2). DNase I protection studies ("footprinting") have shown that T antigen bound to site I protects sequences from approximately nucleotide 5175 to 5215, while site II is protected from nucleotide 5215 to 35 (10, 11). T antigen site II is essential for SV40 replication, since mutations in this region can completely inhibit replication in vivo and in vitro, whereas site I can be removed with less severe effects (12-15). However, many studies examining T antigen interactions with the SV40 ori in vitro were performed under conditions that do not support replicatione.g., incubations were at low temperatures (usually 00C) and lacked ATP.The interactions between T antigen and ori have been examined recently under conditions that support SV40 DNA replication. The presence of ATP or dATP induced formation of a complex that was detected by electron microscopy and gel mobility-shift assays (16). In this paper, we have examined in greater detail the interaction of T antigen with ori in vitro, using DNase I footprinting and nitrocellulose filter binding. We have found that under conditions that support SV40 DNA replication, incubation at 37°C in the presence of ATP greatly stimulated the formation of a complex of T antigen with binding site II. We suggest that the ATPdependent formation of this complex is an essential prerequisite for the initiation of SV40 DNA replication. Similar results have also been obtained by Deb and Tegtmeyer (17). MATERIALS AND METHODSPreparation of T Antigen. T antigen was immunoaffinitypurified from COS-1 cells infected with SV40 cs1085 as described by Wobbe et al. (18).Preparation of End-Labeled SV40 ori Fragments. pSV01-AEP (18) plasmid ori DNA was used to prepare the ori wild-type (wt) fragment (Fig. 1B). The 5'-32P-labeled ori wt fragment was prepared by first cleaving pSV01AEP with EcoRI and, after dephosphorylation, labeling 5' ends with [y-32P]ATP and phage T4 polynucleotide kinase (Boehringer Mannheim). Fragments labeled uniquely on the top strand (the strand containing adenosine residues in positions 21 to 28) were produced by digestion with Sph I, whereas fragments specifically labeled on the bottom strand were prepared by cleavage with the restriction enzyme Hinf I. The digested DNA fragments were separated by electrophoresis on 8% polyacrylamide gels, and the desired fragment was localized by autoradiography and purified.pOR1 DNA (12) was used to prepare DNA fragments containing the SV40 "ori core" (defined in Results; Fig. 1A). ori core fragments, uniquely 5'-end-labeled on the top strand, were obtained by initial cleavage with restriction enzyme Nae I followed by end-labeling and Aha II digestion. The order of the two restriction digestions was reversed to prepare ori core DNA labeled on the bottom strand...

Assembly of T-Antigen Double Hexamers on the Simian Virus 40 Core Origin Requires Only a Subset of the Available Binding Sites

“…DNase I-based footprinting experiments demonstrated that in the presence of ATP, T-ag protected a region that extended over the entire core origin (6,7,22,57,58). The larger size of the DNase I footprint relative to the 1,10-phenanthroline-copper footprint may simply reflect a steric clash between DNase I and T-ag double hexamers.…”

“…Initial studies demonstrated that in the absence of ATP, T-ag formed oligomers on the SV40 origin (8,30,31,55). In subsequent studies, it was observed that the addition of ATP stimulated the binding of T-ag to the core origin 10-to 15-fold and induced the formation of a larger multimeric complex (6,18,22). With electron microscopy, it was determined that the multimeric complex was a bilobed structure (18,19) and that each lobe contained six monomers of T-ag (48,62).…”

Model for T-Antigen-Dependent Melting of the Simian Virus 40 Core Origin Based on Studies of the Interaction of the Beta-Hairpin with DNA

“…Previously described nitrocellulose filter binding assays were used to measure binding of T-ag to the SV40 origin (11,51). Reaction mixtures (20 l) contained 7 mM MgCl 2 , 0.5 mM DTT, 40 mM creatine phosphate (di-Tris salt [pH 7.6]), 0.48 g of creatine phosphate kinase, 4 mM AMP-PNP, 0.2 mg of bovine serum albumin per ml, 0.8 g of HaeIIIdigested pBR322 DNA, 25 fmol (ϳ0.4 ϫ 10 6 cpm/pmol) of the 64-bp core origin or a 64-bp control oligonucleotide derived from the SV40 enhancer (41), and 490 ng (6 pmol) of T-ag or the KH512/513AA double mutant.…”

Initiation of simian virus 40 DNA synthesis in vitro.

“…Binding site II is contained within a region of the origin, termed the core origin, which is necessary and sufficient for SV40 replication both in vitro and in vivo (references 2, 9, 13, 37, and 50 and references therein). Binding of T-Ag to site II is stimulated 10-to 15-fold in the presence of ATP (4,12). Moreover, in the presence of ATP, T-Ag forms a complex nucleoprotein structure at the core origin (11) in which T-Ag is assembled as a double hexamer (32).…”