Assembly of T-antigen double hexamers on the simian virus 40 core origin requires only a subset of the available binding sites

Abstract

Initiation of simian virus 40 (SV40) DNA replication is dependent upon the assembly of two T-antigen (T-ag) hexamers on the SV40 core origin. To further define the oligomerization mechanism, the pentanucleotide requirements for T-ag assembly were investigated. Here, we demonstrate that individual pentanucleotides support hexamer formation, while particular pairs of pentanucleotides suffice for the assembly of T-ag double hexamers. Related studies demonstrate that T-ag double hexamers formed on "active pairs" of pentanucleotides catalyze a set of previously described structural distortions within the core origin. For the four-pentanucleotide-containing wild-type SV40 core origin, footprinting experiments indicate that T-ag double hexamers prefer to bind to pentanucleotides 1 and 3. Collectively, these experiments demonstrate that only two of the four pentanucleotides in the core origin are necessary for T-ag assembly and the induction of structural changes in the core origin. Since all four pentanucleotides in the wild-type origin are necessary for extensive DNA unwinding, we concluded that the second pair of pentanucleotides is required at a step subsequent to the initial assembly process.

FIG. 1

Map of the SV40 origin region and sequences of representative oligonucleotides used in this study. (A) The relative locations of the core origin (extending between nucleotides 5211 and 31) (20), T-ag binding site I, the 21-bp repeats, and a partial copy of one of the 72-bp enhancer elements are shown. Also indicated are the template positions from which the enhancer control oligonucleotide was derived (see below). SV40 nucleotides are numbered as described elsewhere (71). (This map is based on the SV40 origin sequences present in plasmid pSV01ΔEP [75].) (B) Representative oligonucleotides from the different pentanucleotide mutant classes are shown, along with the control oligonucleotides used in this study. Diagram D1 shows the DNA sequence of the 64-bp wild-type core origin. The arrows depict the four GAGGC pentanucleotide recognition sequences for T-ag within site II; the pentanucleotides are numbered as previously described (42). The locations of the AT-rich region and the EP region are also depicted. Diagram D2 shows a representative member of the single-pentanucleotide core origin mutations, 2m. Boldface letters, transition mutations replacing the GAGGC pentanucleotides. Additional members of this class had transition mutations in pentanucleotide 1, 3, or 4. Diagram D3 shows a representative member of the double-pentanucleotide core origin mutations, 2-4m (pentanucleotides 1 and 3 were intact). Five additional double-pentanucleotide mutations were synthesized (1-2, 1-3, 1-4, 2-3, 2-4, and 3-4). Diagram D4 shows a representative member of the triple-pentanucleotide core origin mutations, 2-3-4m. Three additional oligonucleotides, containing intact copies of pentanucleotides 2, 3, and 4, were also synthesized (1-3-4m, 1-2-4m, and 1-2-3m, respectively). Diagram D5 shows the sequence of an oligonucleotide used as a control in certain band shift assays (1-2-3-4m control oligonucleotide). This DNA molecule contained transition mutations in all four pentanucleotides. Diagram D6 shows the 64-bp enhancer control oligonucleotide from the region depicted in panel A. The bar depicts DNA derived from the SV40 enhancer, while the location of a single GAGGC pentanucleotide is indicated by the arrow. The numbers in parentheses are non-SV40 sequences labeled according to the pSV01ΔEP numbering system (75).

FIG. 2

Representative gel mobility assay used to assess T-ag oligomerization on the triple-pentanucleotide mutants. As a positive control, a band shift reaction was conducted with T-ag (∼0.5 μg) and the 64-bp core oligonucleotide (lane 2). Reaction products formed with T-ag and 2-3-4m (pentanucleotide 1 intact), 1-3-4m (pentanucleotide 2 intact), 1-2-4m (pentanucleotide 3 intact), and 1-2-3m (pentanucleotide 4 intact) are presented in lanes 4, 6, 8, and 10, respectively. The products formed with the enhancer control oligonucleotide and T-ag are presented in lane 12. The products of band shift reactions conducted in the absence of T-ag and the indicated oligonucleotides are presented in the odd-numbered lanes. The arrows indicate the positions of T-ag hexamers (H), T-ag double hexamers (DH), free DNA (F), and single-stranded DNA (s.s.) generated as a result of the helicase activity of T-ag (17, 32, 66). The reactions were performed at a T-ag/oligonucleotide ratio of 120:1.

FIG. 3

Representative gel mobility shift assays used to assess T-ag oligomerization on the double-pentanucleotide mutants. (A) This set of reactions was conducted in the presence of the nonhydrolyzable analog of ATP, AMP-PNP. As a positive control, a band shift reaction was conducted with the 64-bp core oligonucleotide and T-ag (lane 2). Reaction products formed with T-ag (∼0.25 μg) and mutants 3-4m (pentanucleotides 1 and 2 intact), 2-4m (pentanucleotides 1 and 3 intact), 1-4m (pentanucleotides 2 and 3 intact), 2-3m (pentanucleotides 1 and 4 intact), 1-3m (pentanucleotides 2 and 4 intact), and 1-2m (pentanucleotides 3 and 4 intact) are presented in lanes 4, 6, 8, 10, 12, and 14, respectively. The products of band shift reactions conducted in the absence of T-ag and the indicated oligonucleotides are presented in the odd-numbered lanes. (B) These reactions are identical to those shown in panel A, except that they were performed in the presence of ATP rather than AMP-PNP. As in panel A, the reactions in the even-numbered lanes were conducted in the presence of T-ag, while those in the odd-numbered lanes were conducted in the absence of T-ag. The even-numbered lanes contained single-stranded (s.s.) DNA generated as a result of the helicase activity of T-ag (17, 32, 66). (C) Double-hexamer assembly in the absence of pentanucleotides was analyzed with the 64-bp 1-2-3-4m control oligonucleotide. As in previous experiments, double-hexamer formation on the 64-bp core oligonucleotide served as a positive control (lanes 2 and 6). Band shift reactions conducted with T-ag and 1-2-3-4m are presented in lanes 4 and 8. The experiments presented in lanes 1 to 4 were conducted in the presence of AMP-PNP, while those in lanes 5 to 8 were conducted in the presence of ATP. Reactions presented in odd-numbered lanes were conducted in the absence of T-ag. The arrows indicate the positions of T-ag hexamers (H), T-ag double hexamers (DH), free DNA (F), and single-stranded DNA (s.s.). Finally, the reactions were conducted at a T-ag/oligonucleotide ratio of 60:1; however, nearly identical results were obtained at a ratio of 120:1.

FIG. 3

Representative gel mobility shift assays used to assess T-ag oligomerization on the double-pentanucleotide mutants. (A) This set of reactions was conducted in the presence of the nonhydrolyzable analog of ATP, AMP-PNP. As a positive control, a band shift reaction was conducted with the 64-bp core oligonucleotide and T-ag (lane 2). Reaction products formed with T-ag (∼0.25 μg) and mutants 3-4m (pentanucleotides 1 and 2 intact), 2-4m (pentanucleotides 1 and 3 intact), 1-4m (pentanucleotides 2 and 3 intact), 2-3m (pentanucleotides 1 and 4 intact), 1-3m (pentanucleotides 2 and 4 intact), and 1-2m (pentanucleotides 3 and 4 intact) are presented in lanes 4, 6, 8, 10, 12, and 14, respectively. The products of band shift reactions conducted in the absence of T-ag and the indicated oligonucleotides are presented in the odd-numbered lanes. (B) These reactions are identical to those shown in panel A, except that they were performed in the presence of ATP rather than AMP-PNP. As in panel A, the reactions in the even-numbered lanes were conducted in the presence of T-ag, while those in the odd-numbered lanes were conducted in the absence of T-ag. The even-numbered lanes contained single-stranded (s.s.) DNA generated as a result of the helicase activity of T-ag (17, 32, 66). (C) Double-hexamer assembly in the absence of pentanucleotides was analyzed with the 64-bp 1-2-3-4m control oligonucleotide. As in previous experiments, double-hexamer formation on the 64-bp core oligonucleotide served as a positive control (lanes 2 and 6). Band shift reactions conducted with T-ag and 1-2-3-4m are presented in lanes 4 and 8. The experiments presented in lanes 1 to 4 were conducted in the presence of AMP-PNP, while those in lanes 5 to 8 were conducted in the presence of ATP. Reactions presented in odd-numbered lanes were conducted in the absence of T-ag. The arrows indicate the positions of T-ag hexamers (H), T-ag double hexamers (DH), free DNA (F), and single-stranded DNA (s.s.). Finally, the reactions were conducted at a T-ag/oligonucleotide ratio of 60:1; however, nearly identical results were obtained at a ratio of 120:1.

FIG. 3

Representative gel mobility shift assays used to assess T-ag oligomerization on the double-pentanucleotide mutants. (A) This set of reactions was conducted in the presence of the nonhydrolyzable analog of ATP, AMP-PNP. As a positive control, a band shift reaction was conducted with the 64-bp core oligonucleotide and T-ag (lane 2). Reaction products formed with T-ag (∼0.25 μg) and mutants 3-4m (pentanucleotides 1 and 2 intact), 2-4m (pentanucleotides 1 and 3 intact), 1-4m (pentanucleotides 2 and 3 intact), 2-3m (pentanucleotides 1 and 4 intact), 1-3m (pentanucleotides 2 and 4 intact), and 1-2m (pentanucleotides 3 and 4 intact) are presented in lanes 4, 6, 8, 10, 12, and 14, respectively. The products of band shift reactions conducted in the absence of T-ag and the indicated oligonucleotides are presented in the odd-numbered lanes. (B) These reactions are identical to those shown in panel A, except that they were performed in the presence of ATP rather than AMP-PNP. As in panel A, the reactions in the even-numbered lanes were conducted in the presence of T-ag, while those in the odd-numbered lanes were conducted in the absence of T-ag. The even-numbered lanes contained single-stranded (s.s.) DNA generated as a result of the helicase activity of T-ag (17, 32, 66). (C) Double-hexamer assembly in the absence of pentanucleotides was analyzed with the 64-bp 1-2-3-4m control oligonucleotide. As in previous experiments, double-hexamer formation on the 64-bp core oligonucleotide served as a positive control (lanes 2 and 6). Band shift reactions conducted with T-ag and 1-2-3-4m are presented in lanes 4 and 8. The experiments presented in lanes 1 to 4 were conducted in the presence of AMP-PNP, while those in lanes 5 to 8 were conducted in the presence of ATP. Reactions presented in odd-numbered lanes were conducted in the absence of T-ag. The arrows indicate the positions of T-ag hexamers (H), T-ag double hexamers (DH), free DNA (F), and single-stranded DNA (s.s.). Finally, the reactions were conducted at a T-ag/oligonucleotide ratio of 60:1; however, nearly identical results were obtained at a ratio of 120:1.

FIG. 4

In situ footprinting of T-ag double-hexamer–core origin complexes with the nuclease activity of 1,10-phenanthroline–copper ion. Lanes 1 and 4 contain the products of control reactions conducted with free DNA isolated from samples containing the 2-4m and 64-bp core oligonucleotides, respectively. Lanes 3 and 6 contain the products of footprinting reactions conducted with the same oligonucleotides but isolated from T-ag double-hexamer complexes. For comparison, lanes 2 and 5 contain the products of footprinting reactions conducted with T-ag-obd_131-260 and the same pair of oligonucleotides. Size markers were generated by subjecting the indicated oligonucleotides to the G and G+A sequencing reactions described by Maxam and Gilbert (49). Flanking each panel is a map of the relative positions of the EP region, pentanucleotides 1 to 4 (arrows), and the AT-rich region. The arrows with the smaller heads represent the complementary sequences of a given pentanucleotide. The experiments were conducted at a 120:1 protein (either T-ag or T-ag-obd_131-260)/oligonucleotide ratio. The oligonucleotides were asymmetrically labeled on the top strands (Fig. 1B).

FIG. 5

Ability of pSV01ΔEP double-pentanucleotide mutants to catalyze T-ag-dependent structural changes in the core origin. T-ag (1 μg) was incubated under replication conditions with pSV01ΔEP(core) (lane 2), pSV01ΔEP(3-4m) (lane 4), pSV01ΔEP(2-4m) (lane 6), pSV01ΔEP(1-4m) (lane 8), pSV01ΔEP (2-3m) (lane 10), pSV01ΔEP(1-3m) (lane 12), pSV01ΔEP(1-2m) (lane 14), or pSV01ΔEP(1-2-3-4m) (lane 16). (In all reactions, the T-ag/plasmid ratio was ∼60:1.) Reactions in odd-numbered lanes were conducted with the indicated plasmids in the absence of T-ag. 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 elements are shown on the right.

FIG. 6

Determination of the relative abilities of the pSV01ΔEP double-pentanucleotide and pSV01ΔEP single-pentanucleotide mutants to support T-ag-dependent unwinding. Samples were analyzed by electrophoreses on a 1.8% agarose gel containing chloroquine (1.5 μg/ml); markers (lanes M) contained restriction fragments generated by cleavage of bacteriophage lambda with HindIII (lane 1; the lower fragment is 2,027 bp and the upper fragment is 2,322 bp) and Form I pSV01ΔEP (lane 2). The topological isomers produced after 15 min of incubation in HeLa cell crude extracts in the presence or absence of T-ag with pSV01ΔEP (lanes 3 and 4), pSV01ΔEP(core) (lanes 5 and 6), pSV01ΔEP(2-4m) (lanes 7 and 8), and pSV01ΔEP(2m) (lanes 9 and 10) are presented. Reactions in lanes 4, 6, 8, and 10 were conducted in the presence of T-ag (2 μg); those in lanes 3, 5, 7, and 9 were conducted in the absence of T-ag. The position of Form U_R, formed in the reaction containing pSV01ΔEP(core), is indicated. The amount of Form U_R generated by pSV01ΔEP(core) is the same as or near the amount produced by pSV01ΔEP. Unwinding reactions were conducted at T-ag/plasmid ratios of 60:1.

FIG. 7

Model depicting T-ag double-hexamer formation on the SV40 core origin. The band shift and 1,10-phenanthroline–copper footprinting experiments indicated that under replication conditions, T-ag double hexamers preferentially occupy pentanucleotides 1 and 3. (The pentanucleotides are symbolized by the arrows below the figure and by bold type in the DNA duplex.) Furthermore, the KMnO₄ footprinting studies demonstrated that double-hexamer formation on pentanucleotides 1 and 3 (as well as pentanucleotides 1 and 4) induces the previously described structural alterations in the AT-rich and EP regions (7, 57). It is assumed that after an initial T-ag monomer interaction with a given pentanucleotide, via T-ag-obd_131-260 (shown in gray), subsequent protein-protein interactions gave rise to mature hexamers.