Sequence requirements for the assembly of simian virus 40 T antigen and the T-antigen origin binding domain on the viral core origin of replication

Abstract

The regions of the simian virus 40 (SV40) core origin that are required for stable assembly of virally encoded T antigen (T-ag) and the T-ag origin binding domain (T-ag-obd(131-260)) have been determined. Binding of the purified T-ag-obd(131-260) is mediated by interactions with the central region of the core origin, site II. In contrast, T-ag binding and hexamer assembly requires a larger region of the core origin that includes both site II and an additional fragment of DNA that may be positioned on either side of site II. These studies indicate that in the context of T-ag, the origin binding domain can engage the pentanucleotides in site II only if a second region of T-ag interacts with one of the flanking sequences. The requirements for T-ag double-hexamer assembly are complex; the nucleotide cofactor present in the reaction modulates the sequence requirements for oligomerization. Nevertheless, these experiments provide additional evidence that only a subset of the SV40 core origin is required for assembly of T-ag double hexamers.

FIG. 1

Sequences of the site II-based oligonucleotides. Diagrams 1 and 3 depict oligonucleotides containing site II, while diagrams 2 and 4 represent control oligonucleotides. The arrows depict the four GAGGC pentanucleotides within site II that are recognition sequences for T-ag, numbered as previously described (43). The names of the oligonucleotides are given to the right of each figure. Lowercase boldface letters in the control oligonucleotides represent the transition mutations (m) introduced into individual GAGGC pentanucleotides. SV40 sequences are numbered as described elsewhere (79).

FIG. 2

Representative gel mobility shift assay used to establish the minimal sequence requirement for T-ag-obd_131–260 binding to site II. Lanes: 2 and 3, products of band shift assays conducted with the 64-bp core oligonucleotide and 3 or 6 pmol, respectively, of T-ag-obd_131–260; 5 and 6, products of band shift assays conducted with the 31-bp site II oligonucleotide and 3 or 6 pmol, respectively, of T-ag-obd_131–260; 8 and 9, products of band shift assays conducted with the 23-bp site II oligonucleotide and 3 or 6 pmol, respectively, of T-ag-obd_131–260. As a control for nonspecific binding, band shift assays were conducted with the 31-bp site IIm control oligonucleotide and either 3 or 6 pmol of T-ag-obd_131–260 (lanes 11 and 12). Lanes 1, 4, 7, and 10 contain the products of band shift assays conducted in the absence of protein with the indicated oligonucleotides. The input or free duplex DNA (F) is indicated by the arrow. The protein-to-oligonucleotide ratios with 3 and 6 pmol of T-ag-obd_131–260 and 25 fmol of oligonucleotide are 120:1 and 240:1, respectively.

FIG. 3

Sequences of the 64-bp core oligonucleotide and mutant forms of this molecule. The locations of the AT-rich regions, site II, and the EP regions are depicted. As in Fig. 1, the arrows depict the four GAGGC pentanucleotides within site II that serve as binding sites for T-ag. Lowercase boldface letters represent transition mutations (m) introduced into the indicated regions. The names of the individual oligonucleotides are given to the right of their sequences.

FIG. 4

Gel mobility shift assays used to establish whether mutant forms of the flanking sequences influence T-ag-obd_131–260 binding to site II. As a positive control, gel mobility shift assays were conducted with the 64-bp core oligonucleotide and either 3 or 6 pmol of T-ag-obd_131–260 (lanes 2 and 3). In related reactions, T-ag-obd_131–260 (3 or 6 pmol) was incubated with the 64-bp EPm (lanes 5 and 6), the 64-bp ATm (lanes 8 and 9), the 64-bp ATm + EPm (lanes 11 and 12), or the 64-bp enhancer control (lanes 14 and 15). The reactions in lanes 1, 4, 7, 10, and 13 were conducted in the absence of protein. The input or free duplex DNA (F) is indicated by the arrow. The protein-to-oligonucleotide ratios used in these reactions are given in the legend to Fig. 2. Quantitation with a PhosphorImager revealed that with 6 pmol of T-ag-obd_131–260, the percentage of input DNA shifted into the major band shift product formed with the 64-bp core, 64-bp EPm, 64-bp ATm, and 64-bp ATm + EPm oligonucleotides was 22.4, 25.7, 24.0, and 23.1%, respectively.

FIG. 5

Determination of whether purified forms of T-ag-obd catalyze structural changes in the core origin. As controls, the SV40 origin-containing plasmid pSV01ΔEP was incubated under replication conditions in the absence (lane 1) or presence (lane 6) of T-ag. The reactions in lanes 2, 3, and 4 were conducted in the presence of the indicated amounts of T-ag-obd_131–260, while the reaction in lane 5 was conducted in the presence of 6 pmol of T-ag-obd_112–260 (39). After treatment with KMnO₄, the sites of oxidation were probed by primer extension reactions with ³²P-labeled oligonucleotide 1 (see Materials and Methods). The primer extension products were analyzed by electrophoresis on a 7% polyacrylamide gel containing 8 M urea. The locations of the EP, site II, and AT-rich sequence elements are indicated on the right.

FIG. 6

Gel mobility shift assay used to establish whether T-ag can assemble on the 31-bp site II oligonucleotide. As a positive control, 3 or 6 pmol of T-ag was incubated with the 64-bp core oligonucleotide (lanes 2 and 3) in the presence of ATP. T-ag (3 or 6 pmol) was also incubated with 25 fmol of the 31-bp site II oligonucleotide in the presence of different analogs of ATP; the reactions in lanes 5 and 6 were conducted in the presence of ATP, those in lanes 8 and 9 were conducted in the presence of AMP-PNP, and those in lanes 11 and 12 were conducted in the presence of ADP. The reactions in lanes 1, 4, 7, and 10 were conducted in the absence of protein. The input or free duplex DNA (F) is indicated by the arrow. Single-stranded DNA (s.s.), the product of the T-ag helicase activity, is present in elevated amounts in lanes 2 and 3. DH, double hexamer; H, hexamer. As in previous examples, the protein-to-oligonucleotide ratios used in these reactions are given in the legend to Fig. 2.

FIG. 7

Filter binding assays used to measure the ability of T-ag to interact with the 31-bp site II oligonucleotide under replication conditions. The interaction of T-ag (0, 3, or 6 pmol) with 25 fmol of the 31-bp site II oligonucleotide was measured by nitrocellulose filter binding assays in the presence of the indicated nucleotide cofactors. The percentage of input oligonucleotide bound to a given filter was determined by scintillation counting. As a positive control, the interaction of T-ag (0, 3, or 6 pmol) with the 64-bp core oligonucleotide was measured in the presence of ATP. Additional controls were conducted with the 64-bp enhancer control and the 31-bp site IIm and the indicated nucleotide cofactors.

FIG. 8

The set of oligonucleotides collectively termed the asymmetric extensions of site II; the names of the individual oligonucleotides are given to the right of their sequences. The locations of the AT-rich region, site II, and the EP region are depicted. As in previous examples, the arrows depict the four GAGGC pentanucleotides within site II that serve as recognition sites for T-ag. Diagram 1 presents the sequence of the oligonucleotide containing site II and the EP region, while diagram 4 presents the sequence of the oligonucleotide-containing site II and the AT region. Control oligonucleotides, containing transition mutations in the EP and AT regions, are depicted in diagrams 2 and 5, respectively. Additional control oligonucleotides, containing the wild-type EP and AT-rich regions and transition mutations in the pentanucleotides are depicted in diagrams 3 and 6, respectively. As in previous examples, lowercase boldface letters represent transition mutations (m) introduced into the indicated regions. Finally, the sequence of the 47-bp control oligonucleotide, used to measure non-sequence-specific binding, is presented in diagram 7.

FIG. 9

Representative gel mobility shift assays used to assess the ability of T-ag to interact with oligonucleotides containing site II and either of the flanking sequences. (A) The experiments were performed in the presence of AMP-PNP with 6 pmol of T-ag and 25 fmol of the indicated oligonucleotide. As a positive control, the reaction in lane 2 was conducted with T-ag and the 64-bp core oligonucleotide. Reaction products formed with T-ag and the 48-bp site II + EP, the 48-bp site II + EPm, and the 48-bp site IIm + EP oligonucleotides are shown in lanes 4, 6, and 8, respectively. Reactions conducted with the 47-bp site II + AT, the 47-bp site II + ATm, and the 47-bp site IIm + AT oligonucleotides are shown in lanes 10, 12, and 14, respectively. The products of band shift reactions conducted in the absence of T-ag and the indicated oligonucleotides are shown in the odd-numbered lanes. (B) The experiments are identical to those in panel A, except that they were conducted in the presence of ATP. In both panels the arrows indicate the positions of T-ag hexamers (H), T-ag double hexamers (DH), and free DNA (F). Single-stranded DNA (s.s.), formed owing to the helicase activity of T-ag, is present in lane 2. The protein-to-oligonucleotide ratio with 6 pmol of T-ag and 25 fmol of oligonucleotide is 240:1.

FIG. 10

Filter binding assays used to measure the ability of T-ag to interact with the asymmetric extensions of site II set of oligonucleotides. The amount of oligonucleotide bound to T-ag was established by nitrocellulose filter binding assays and scintillation counting. The interactions of T-ag (0, 3, and 6 pmol) with these oligonucleotides were measured in the presence of ATP (A), AMP-PNP (B), or ADP (C) and in the absence of nucleotide (D). The names of the individual oligonucleotides are shown to the right of the figure.

FIG. 11

Model depicting the requirements for binding of T-ag-obd_131–260 and T-ag to site II and the asymmetric extensions of site II. (A) A slightly elongated version of site II is necessary and sufficient for binding of T-ag-obd_131–260 (abbreviated T-obd in this model). The GAGGC pentanucleotides are shown in bold and numbered as previously described (43). Based on results obtained in a previous study (39), T-ag-obd_131–260 is depicted bound to pentanucleotides 1 and 3 as a dimer (B). In contrast, T-ag and therefore the T-ag-obd present in this molecule cannot bind to oligonucleotides containing just site II. The arrows covering T-ag-obd are used to symbolize that in the context of T-ag, this domain cannot bind to site II. (C) In the presence of either of the flanking sequences, the obstacle(s) to the interactions of T-ag with site II are removed. It is proposed that the flanking sequences enable additional T-ag/origin contacts, depicted by the extended arrows, and that these interactions result in conformational changes that expose T-ag-obd. (D) Upon binding of a T-ag monomer to a pentanucleotide, protein-protein interactions give rise to hexamers; in turn, hexamers promote the cooperative assembly of double hexamers on active pairs of pentanucleotides. The bold arrow is used to show that assembly events involving the EP region are preferred over those involving the AT-rich region (see the text). Finally, while subfragments of the core origin support double-hexamer formation in the presence of AMP-PNP or ADP, only hexamers are detected in the presence of ATP. Therefore, ATP or ATP hydrolysis expands the repertoire of T-ag/core interactions necessary for double-hexamer formation or stability.