The simian virus 40 core origin contains two separate sequence modules that support T-antigen double-hexamer assembly

Abstract

Using subfragments of the simian virus 40 (SV40) core origin, we demonstrate that two alternative modules exist for the assembly of T-antigen (T-ag) double hexamers. Pentanucleotides 1 and 3 and the early palindrome (EP) constitute one assembly unit, while pentanucleotides 2 and 4 and the AT-rich region constitute a second, relatively weak, assembly unit. Related studies indicate that on the unit made up of pentanucleotide 1 and 3 and the EP assembly unit, the first hexamer forms on pentanucleotide 1 and that owing to additional protein-DNA and protein-protein interactions, the second hexamer is able to form on pentanucleotide 3. Oligomerization on the unit made up of pentanucleotide 2 and 4 and the AT-rich region is initiated by assembly of a hexamer on pentanucleotide 4; subsequent formation of the second hexamer takes place on pentanucleotide 2. Given that oligomerization on the SV40 origin is limited to double-hexamer formation, it is likely that only a single module is used for the initial assembly of T-ag double hexamers. Finally, we discuss the evidence that nucleotide hydrolysis is required for the remodeling events that result in the utilization of the second assembly unit.

FIG. 1

Sequences of the 64-bp core oligonucleotide and a set of oligonucleotides derived from the site II + EP oligonucleotide. (A) The 64-bp core origin; locations of the AT, site II, and the EP regions are indicated. Arrows depict the four GAGGC pentanucleotides within site II that serve as recognition sites for T-ag; pentanucleotides are numbered as previously described (38). SV40 sequences are numbered as described elsewhere (72). (B) Sequences of a set of oligonucleotides based on the 48-bp site II + EP oligonucleotide. The sequence of the 48-bp site II + EP oligonucleotide is presented in diagram 1. Diagram D2 presents the sequence of the 48-bp penta 1, 3 + EP oligonucleotide, a representative member of the two pentanucleotide + EP set of oligonucleotides. During the synthesis of the 48-bp penta 1, 3 + EP oligonucleotide, transition mutations were introduced at pentanucleotides 2 and 4. These mutations are indicated by the lowercase bold letters. Although not depicted, the 48-bp penta 1, 2 + EP, penta 1, 4 + EP, penta 2, 3 + EP, penta 2, 4 + EP, and penta 3, 4 + EP oligonucleotides were also synthesized. Diagram D3 presents the sequence of the 48-bp penta 1, 3 + EPm oligonucleotide. This oligonucleotide is similar to the 48-bp penta 1, 3 + EP oligonucleotide except that additional transition mutations have replaced the EP region (symbolized by lowercase bold letters). A representative member of the single pentanucleotide + EP set of oligonucleotides, the 48-bp penta 1 + EP oligonucleotide, is presented in diagram 4. During synthesis of this oligonucleotide, transition mutations were introduced at pentanucleotides 2, 3, and 4 (indicated by lowercase bold letters). The sequence of the 48-bp penta 1 + EPm oligonucleotide is presented in diagram 5; this molecule is similar to the 48-bp penta 1 + EP oligonucleotide except that additional transition mutations have replaced the EP region (indicated by lowercase bold letters). Although not depicted, the 48-bp penta 3 + EP and 48-bp penta 3 + EPm oligonucleotides were also synthesized. (C) Finally, the sequence of the 47-bp control oligonucleotide is presented; this molecule served as a control for non-sequence-specific binding events.

FIG. 2

Representative gel mobility shift assay used to determine the sequence requirements for double hexamer formation on the two pentanucleotide + EP-based set of oligonucleotides. (A) Experiments were performed in the presence of AMP-PNP with 6 pmol of T-ag and 25 fmol of the indicated oligonucleotide. As positive controls, the reaction displayed in lane 2 was conducted with the 64-bp core oligonucleotide while that displayed in lane 4 was performed with the 48-bp site II + EP oligonucleotide. Reactions displayed in lanes 6, 8, 10, 12, 14, and 16 were conducted with the indicated members of the two pentanucleotide + EP set of oligonucleotides. As a negative control, the reaction in lane 18 was conducted with the 47-bp control oligonucleotide. Reactions in the odd-numbered lanes were conducted with the indicated oligonucleotides in the absence of T-ag. The arrows indicate the positions of T-ag hexamers (H) and T-ag double hexamers (DH). The position of input or free DNA is indicated by a bracket. (B) The reactions in Fig. 2A and similar reactions conducted in the presence of ADP, ATP, and no exogenous nucleotides (data not shown) were quantitated with a Molecular Dynamics PhosphorImager in order to determine the percentage of input DNA shifted into double hexamers. The nucleotide cofactor used in a given reaction is shown to the right of the figure, and the names of the individual oligonucleotides are presented along the x axis, while the percentage of input DNA present in double hexamers is listed on the y axis.

FIG. 3

Representative gel mobility shift assay used to establish which components of the 48-bp penta 1, 3 + EP assembly unit are required for hexamer and double-hexamer formation. (A) Reactions were conducted in the presence of AMP-PNP with 6 pmol of T-ag and 25 fmol of the indicated oligonucleotide. As a positive control, one reaction was conducted with the 48-bp penta 1, 3 + EP oligonucleotide (lane 2). To test the role of the EP in these assembly events, a reaction was conducted with the 48-bp penta 1, 3 + EPm oligonucleotide (lane 4). Reactions conducted with the single pentanucleotide containing 48-bp penta 1 + EP and 48-bp penta 1 + EPm oligonucleotides are presented in lanes 6 and 8, respectively. Similar reactions, conducted with the 48-bp penta 3 + EP and 48-bp penta 3 + EPm oligonucleotides, are presented in lanes 10 and 12. To assay for non-sequence-specific binding events, a reaction was conducted with the 47-bp control oligonucleotide (lane 14). The reactions in the odd-numbered lanes were conducted in the absence of protein. The positions of T-ag hexamers (H) and double hexamers (DH) are indicated by arrows. The location of input or free DNA is indicated by a bracket. (B) The reactions in Fig. 3A and similar reactions conducted in the presence of ADP, ATP, and no exogenous nucleotides (data not shown) were quantitated with a Molecular Dynamics Phosphor- Imager. The percentage of input DNA, containing single pentanucleotides, that was shifted into hexamers is presented in histogram 2. The nucleotide cofactor used in a given set of reactions is shown to the right of the figure, and the names of the individual oligonucleotides are presented along the x axis, while the percentage of input DNA present in the hexamer species is listed on the y axis. Histogram 1 indicates the quantitative impact of mutating the EP region on T-ag oligomerization into hexamers and double hexamers.

FIG. 4

Sequences of the site II + AT-based oligonucleotides. Diagram D1 provides the sequence of the 47-bp site II + AT oligonucleotide. As in previous examples, the arrows depict the four GAGGC pentanucleotides that serve as recognition sites for T-ag. A representative member of the two pentanucleotide + AT set of oligonucleotides is presented in diagram 2. During the synthesis of the 47-bp penta 2, 4 + AT oligonucleotide, transition mutations were introduced at pentanucleotides 1 and 3. These mutations are indicated by lowercase bold letters. Although not depicted, the 47-bp penta 1, 2 + AT, penta 1, 3 + AT, penta 1, 4 + AT, penta 2, 3 + AT, and penta 3, 4 + AT oligonucleotides were also synthesized. Diagram D3 presents the sequence of the 47-bp penta 2, 4 + ATm oligonucleotide; this molecule is similar to the 47-bp penta 2, 4 + AT oligonucleotide except that additional transition mutations have replaced the AT (indicated by the lowercase bold letters). A representative member of the single pentanucleotide + AT set of oligonucleotides, the 47-bp penta 4 + AT oligonucleotide, is presented in diagram 4. During the synthesis of the 47-bp penta 4 + AT oligonucleotide, transition mutations were introduced at pentanucleotides 1, 2, and 3 (indicated by the lowercase bold letters). Diagram D5 presents the 47-bp penta 4 + ATm oligonucleotide; this molecule is similar to the 47-bp penta 4 + AT oligonucleotide except that the AT-rich region has been replaced by additional transition mutations (indicated by lowercase bold letters). Finally, although not depicted, the 47-bp penta 2 + AT and 47-bp penta 2 + ATm oligonucleotides were also synthesized.

FIG. 5

Representative gel mobility shift assay used to determine the sequence requirements for double hexamer formation on the two pentanucleotide + AT-based set of oligonucleotides. (A) Experiments were performed in the presence of AMP-PNP with 6 pmol of T-ag and 25 fmol of the indicated oligonucleotide. As positive controls, reactions were conducted with the 64-bp core oligonucleotide (lane 2) and the 47-bp site II + AT oligonucleotide (lane 4). The reactions displayed in lanes 6, 8, 10, 12, 14, and 16 were conducted with the indicated members of the two pentanucleotide + AT set of oligonucleotides. The reaction shown in lane 18 was conducted with the 47-bp control oligonucleotide. Reactions in the odd-numbered lanes were conducted in the absence of T-ag. The arrows indicate the positions of T-ag hexamers (H) and double hexamers (DH). The position of input or free DNA is indicated by a bracket. (B) The reactions shown in Fig. 5A and similar reactions conducted in the presence of ADP, ATP, and no exogenous nucleotides (data not shown) were quantitated with a Molecular Dynamics PhosphorImager to determine the percentage of input DNA shifted into double hexamers. The nucleotide cofactor is indicated to the right of the figure, and the names of the individual oligonucleotides are presented along the x axis, while the percentage of input DNA present in the double hexamers is indicated on the y axis.

FIG. 6

Representative gel mobility shift assay used to establish which components of the 47-bp penta 2, 4 + AT assembly unit are required for hexamer and double-hexamer formation. (A) Reactions were conducted in the presence of AMP-PNP with 6 pmol of T-ag and 25 fmol of the indicated oligonucleotide. As a positive control, one reaction was conducted with the 47-bp penta 2, 4 + AT oligonucleotide (lane 2). To test the role of the AT in these assembly events, a reaction was conducted with the 47-bp penta 2, 4 + ATm oligonucleotide (lane 4). Reactions conducted with the single pentanucleotide containing 47-bp penta 4 + AT and 47-bp penta 4 + ATm oligonucleotides are presented in lanes 6 and 8, respectively. Similar reactions, conducted with the 47-bp 2 + AT and 47-bp penta 2 + ATm oligonucleotides, are presented in lanes 10 and 12, respectively. To assay for non-sequence-specific binding events, an additional reaction was conducted with the 47-bp control oligonucleotide (lane 14). Reactions in the odd-numbered lanes were conducted in the absence of protein. The positions of T-ag hexamers (H) and double hexamers (DH) are indicated by arrows, while the position of free DNA is indicated by a bracket. (B) The reactions shown in Fig. 6A and similar reactions conducted in the presence of ADP, ATP, and no exogenous nucleotides (data not shown) were quantitated with a Molecular Dynamics PhosphorImager. Histogram 2 displays the percentage of input DNA, containing single pentanucleotides, that is shifted into hexamers. The nucleotide cofactor used in a given set of reactions is shown to the right of the figure, the names of the individual oligonucleotides are shown on the x axis, and the percentage of input DNA shifted into hexamers is shown on the y axis. Histogram 1 reveals the quantitative impact of mutating the AT on T-ag oligomerization on the penta 2, 4 + AT assembly unit.

FIG. 7

Filter binding assays used to measure the ability of T-ag to bind to oligonucleotides derived from the site II + EP- and site II + AT-based oligonucleotides. (A) Reactions were performed in the presence of AMP-PNP and the indicated amounts of T-ag (0, 3, and 6 pmol). The percentage of oligonucleotide bound to T-ag was established by nitrocellulose filter binding assays and scintillation counting. (B) The same set of oligonucleotides were used in additional nitrocellulose filter binding assays conducted in the presence of ATP. Following the addition of T-ag (0, 3, 6 pmol), the amount of bound oligonucleotide was determined. It is noted that a larger percentage of the substrates are bound in the filter binding assays than in the gel shift assays (compare the data in Fig. 7 with those in Fig. 3B and 6B). This may reflect that the T-ag–DNA complexes are subjected to harsher conditions during gel electrophoresis than during filter binding.

FIG. 8

A model illustrating the relative positions of the penta 1, 3 + EP and penta 2, 4 + AT assembly units on the core origin and the formation of hexamers and double hexamers on oligonucleotides containing single assembly sites. (A) To depict the relative positions of the penta 1, 3 + EP and penta 2, 4 + AT assembly units, the 64-bp core origin is shown as a B DNA helix. The penta 1, 3 + EP assembly unit is shown in green, while the penta 2, 4 + AT assembly unit is shown in yellow. The locations of individual GAGGC pentanucleotides are indicated by arrows; the positions of the flanking sequences are also indicated. (B) Models for T-ag assembly events on the penta 2, 4 + AT (left) and 1, 3 + EP (right) assembly units (line 1). The structures used to depict T-ag monomers and hexamers are based on transmission electron microscopy studies reported by Valle et al. (73). Smaller circles represent the T-ag-obd, while the remaining residues of T-ag are represented by larger circles. Pentanucleotides proximal to the flanking sequences are recognized by the T-ag-obd, while non-T-ag-obd residues make both sequence-specific and non-sequence-specific interactions with the flanking sequences (this study; reference and references therein). Following monomer binding (line 2), protein-protein interactions give rise to hexamer formation (line 3). Hexamer formation enables additional protein-protein and protein-DNA interactions to take place and subsequent formation of double hexamers to occur (line 4). Boldface arrows show that assembly on the penta 1, 3 + EP assembly unit is preferred over the penta 2, 4 + AT assembly unit.