Four major sequence elements of simian virus 40 large T antigen coordinate its specific and nonspecific DNA binding.

By mutational analysis, we have identified a motif critical to the proper recognition and binding of simian virus 40 large tumor antigen (T antigen) to virus DNA sequences at the origin of DNA replication. This motif is tripartite and consists of two elements (termed A1 and B2) that are necessary for sequence-specific binding of the origin and a central element (B1) which is required for nonspecific DNA-binding activity. Certain amino acids in elements A1 (residues 152 to 155) and B2 (203 to 207) may make direct contact with the GAGGC pentanucleotide sequences in binding sites I and II on the DNA. Alternatively, these two elements could determine the proper structure of the DNA-binding domain, although for a number of reasons we favor the first possibility. In contrast, element B1 (183 to 187) is most likely important for recognizing a general structural feature of DNA. Elements A1 and B2 are nearly identical in all known papovavirus T antigens, whereas B1 is identical only in the closely related papovaviruses simian virus 40, BK virus, and JC virus. In addition to these three elements, a fourth (B3; residues 215 to 219) is necessary for the binding of T antigen to site II but not to site I. We propose that additional contact sites on T antigen are involved in the interaction with site II to initiate the replication of the viral DNA.     

Topography of simian virus 40 A protein-DNA complexes: arrangement of pentanucleotide interaction sites at the origin of replication.

Investigation of the DNA binding properties of the simian virus 40 (SV40) A protein (large T antigen) and the hybrid adenovirus-SV40 D2 protein revealed that both viral proteins protect similar regions of SV40 DNA from digestion by DNase I or methylation by dimethyl sulfate. However, the interaction of D2 protein with DNA was more sensitive to increases of NaCl concentration than was the interaction of wild-type SV40 A protein. Dimethylsulfate footprinting identified 13 DNA pentanucleotide contact sites at the viral origin of replication. The sequences of these sites corresponded to the consensus family 5'-(G greater than T) (A greater than G)GGC-3'. The pentanucleotides were distributed in three regions of origin DNA. Region I contained three pentanucleotide contact sites arranged as direct repetitions encompassing a span of 23 base pairs. In region II, four pentanucleotides were oriented as inverted repetitions that also spanned a total of 23 base pairs. Region III had six recognition pentanucleotides arranged as direct repetitions in a space of 59 base pairs. These fundamental variations in DNA arrangement are likely to determine different patterns of protein binding in each region.     

Preferential binding of simian virus 40 T-antigen dimers to origin region I.

The sequence components that direct high-affinity binding of simian virus 40 (SV40) T antigen to SV40 origin region I are composed of two recognition pentanucleotides separated by a spacer. This region has binding sites for two T-antigen monomeric units. We extended the tripartite region I sequence by one and two sets of spacers and pentanucleotides and also shortened the region by one pentanucleotide. Our T-antigen-binding studies with these constructs show that the protein has a strong preference for binding to an even rather than an odd number of pentanucleotides separated by spacer sequences. Gel retardation assays reveal that the size of the complex formed between the 17-base-pair region I sequence and T antigen did not increase when the sequence was extended with one spacer-pentanucleotide sequence but did increase with two such units. DNase I footprinting and fragment assay experiments indicate that the protein did not protect a pentanucleotide that was not paired with another pentanucleotide. The unpaired pentanucleotide resumed its binding activity when it was paired with a spacer and another pentanucleotide sequence. We propose that T antigen binds to region I as a preformed dimer.     

Three domains in the simian virus 40 core origin orchestrate the binding, melting, and DNA helicase activities of T antigen.

The simian virus 40 (SV40) core origin of replication consists of three functional domains. The sequence 5'-CACTACTTCTGGAATAG-3' with an imperfect inverted repeat (underlined), a palindrome with four 5'-GAGGC-3' pentanucleotide repeats, and a 17-base-pair A + T-rich segment. We have been able to assign primary functions to each domain. Remarkably, SV40 large T antigen melted the inverted repeat domain in the complete absence of other origin sequences. Presumably, this protein-DNA interaction initiates a replication bubble that leads to daughter strand DNA synthesis. The pentanucleotide domain alone docked and arranged T antigen at the origin. The A + T-rich domain had no independent function, but, in the presence of the other two domains, allowed bound T antigen to extend the replication bubble. Thus, three domains of the origin coordinate the binding, melting, and DNA helicase activities of T antigen in an ordered sequence of events to initiate DNA replication.     

The T-antigen-binding domain of the simian virus 40 core origin of replication.

The simian virus 40 origin of replication contains a 27-base-pair palindrome with the sequence 5'-CA-GAGGC-C-GAGGC-G-GCCTC-G-GCCTC-TG-3'. The four 5'-GAGGC-3'/5'-GCCTC-3' pentanucleotides are known contact sites for simian virus 40 T-antigen binding in vitro. We used oligonucleotide-directed cassette mutagenesis to identify features of this palindrome that are important for the initiation of DNA replication in vivo. Each base pair of a pentanucleotide is crucial for DNA replication. In contrast, sequences adjacent to pentanucleotides have little or no effect on replication. Thus, the pentanucleotide is the basic functional unit, not only for T-antigen binding but also for DNA replication. All four pentanucleotides are indispensable in the initiation process. The spacing of pentanucleotides is crucial because duplication of the single base pair between binding sites has a far greater effect on replication than does substitution of the same base pair. Inversion of any pentanucleotide blocks DNA synthesis. Thus, the pentanucleotide is not a functionally symmetrical unit. We propose that each pentanucleotide positions a monomer of T antigen at the proper distance, rotation, and orientation relative to other T-antigen monomers and to other origin domains and that such positioning leads to subsequent events in replication.     

Topography of simian virus 40 A protein-DNA complexes: arrangement of protein bound to the origin of replication.

DNA binding regions I, II, and III at the origin of replication have different arrangements of A protein (T antigen) recognition pentanucleotides. The A protein also protects each region from DNase in distinctly different patterns. Footprint and fragment assays led to the following conclusions: (i) in some cases a single recognition pentanucleotide is sufficient to direct the binding and accurate alignment of A protein on DNA; (ii) the A protein binds within isolated region I or II in a sequential process leading to multiple overlapping areas of DNase protection within each region; and (iii) the 23-base pair span of recognition sequences in region II allows binding and protection of a longer length of DNA than the 23-base pair span in region I. We propose a model of protein binding that addresses the problem of variations in the arrangement of pentanucleotides in regions I and II and explains the observed DNase protection patterns. The central feature of the model requires each protomer of A protein to bind to a pentanucleotide in a unique direction. The resulting orientation of protein would protect more DNA at the 5' end of the 5'-GAGGC-3' recognition sequence than at the 3' end. The arrangement of multiple protomers at the origin of simian virus 40 replication is discussed.     

Identification of simian virus 40 T-antigen residues important for specific and nonspecific binding to DNA and for helicase activity.

We have previously identified three regions (called elements) in the DNA-binding domain of simian virus 40 large tumor (T) antigen which are critical for binding of the protein to the recognition pentanucleotides GAGGC at the viral replication origin. These are elements A (residues 147 to 159), B1 (185 to 187), and B2 (203 to 207). In this study, we generated mutants of simian virus 40 in order to make single-point substitution mutations at nearly every site in these three elements. Each mutation was tested for its effect on virus replication, and T antigen was produced from all replication-negative mutants. The mutant proteins were assayed for binding to several different DNA substrates and for helicase activity. We found that within each element, mutations at some sites had major effects on DNA binding while mutations at other sites had moderate, mild, or minimal effects, suggesting that some residues are more important than others in mediating DNA binding. Furthermore, we provide evidence that certain residues in elements A and B2 (Ala-149, Phe-159, and His-203) participate in nonspecific double-stranded and helicase substrate (single-stranded) DNA binding while others (Ser-147, Ser-152, Asn-153, Thr-155, Arg-204, Val-205, and Ala-207) are involved in sequence-specific binding at the origin. The residues in element B1 (primarily Ser-185 and His-187) take part only in nonspecific DNA binding. The amino acids important for nonspecific DNA binding are also required for helicase activity, and we hypothesize that they make contact with the sugar-phosphate backbone of DNA. On the other hand, those involved in sequence-specific binding are not needed for helicase activity. Finally, our analysis showed that three residues (Asn-153 and Thr-155 in element A and Arg-204 in element B2) may be the most important for sequence-specific binding. They are likely to make direct or indirect contacts with the pentanucleotide sequences at the origin.     

Simian virus 40 and polyomavirus large tumor antigens have different requirements for high-affinity sequence-specific DNA binding.

By using a DNA fragment immunoassay, the binding of simian virus 40 (SV40) and polyomavirus (Py) large tumor (T) antigens to regulatory regions at both viral origins of replication was examined. Although both Py T antigen and SV40 T antigen bind to multiple discrete regions on their proper origins and the reciprocal origin, several striking differences were observed. Py T antigen bound efficiently to three regions on Py DNA centered around an MboII site at nucleotide 45 (region A), a BglI site at nucleotide 92 (region B), and another MboII site at nucleotide 132 (region C). Region A is adjacent to the viral replication origin, and region C coincides with the major early mRNA cap site. Weak binding by Py T antigen to the origin palindrome centered at nucleotide 3 also was observed. SV40 T antigen binds strongly to Py regions A and B but only weakly to region C. This weak binding on region C was surprising because this region contains four tandem repeats of GPuGGC, the canonical pentanucleotide sequence thought to be involved in specific binding by T antigens. On SV40 DNA, SV40 T antigen displayed its characteristic hierarchy of affinities, binding most efficiently to site 1 and less efficiently to site 2. Binding to site 3 was undetectable under these conditions. In contrast, Py T antigen, despite an overall relative reduction of affinity for SV40 DNA, binds equally to fragments containing each of the three SV40 binding sites. Py T antigen, but not SV40 T antigen, also bound specifically to a region of human Alu DNA which bears a remarkable homology to SV40 site 1. However, both tumor antigens fail to precipitate DNA from the same region which has two direct repeats of GAGGC. These results indicate that despite similarities in protein structure and DNA sequence, requirements of the two T antigens for pentanucleotide configuration and neighboring sequence environment are different.     

Sequence-specific binding of simian virus 40 A protein to nonorigin and cellular DNA.

The simian virus 40 A protein (T antigen) recognized and bound to the consensus sequence 5'-GAGGC-3' in DNA from many sources. Sequence-specific binding to single pentanucleotides in randomly chosen DNA predominated over binding to nonspecific sequences. The asymmetric orientation of protein bound to nonorigin recognition sequences also resembled that of protein bound to the origin region of simian virus 40 DNA. Sequence variations in the DNA adjacent to single pentanucleotides influenced binding affinities even though methylation interference and protection studies did not reveal specific interactions outside of pentanucleotides. Thus, potential locations of A protein bound to any DNA can be predicted although the determinants of binding affinity are not yet understood. Sequence-specific binding of A protein to cellular DNA would provide a mechanism for specific alterations of host gene expression that facilitate viral function.     

Mechanisms of simian virus 40 T-antigen activation by phosphorylation of threonine 124.

Previous studies have shown that phosphorylation of simian virus 40 (SV40) T antigen at threonine 124 enhances the binding of T antigen to the SV40 core origin of replication and the unwinding of the core origin DNA via hexamer-hexamer interactions. Here, we report that threonine 124 phosphorylation enhances the interaction of T-antigen amino acids 1 to 259 and 89 to 259 with the core origin of replication. Phosphorylation, therefore, activates the minimal DNA binding domain of T antigen even in the absence of domains required for hexamer formation. Activation is mediated by only one of three DNA binding elements in the minimal DNA binding domain of T antigen. This element, including amino acids 167, 215, and 219, enhances binding to the unique arrangement of four pentanucleotides in the core origin but not to other pentanucleotide arrangements found in ancillary regions of the SV40 origin of replication. Interestingly, the same four pentanucleotides in the core origin are necessary and sufficient for phosphorylation-enhanced DNA binding. Further, we show that phosphorylation of threonine 124 promotes the assembly of high-order complexes of the minimal DNA binding domain of T antigen with core origin DNA. We propose that phosphorylation induces conformational shifts in the minimal DNA binding domain of T antigen and thereby enhances interactions among T-antigen subunits oriented by core origin pentanucleotides. Similar subunit interactions would enhance both assembly of full-length T antigen into binary hexamer complexes and origin unwinding.     

The zinc finger region of simian virus 40 large T antigen is needed for hexamer assembly and origin melting.

Simian virus 40 large T antigen contains a single sequence element with an arrangement of cysteines and histidines that is characteristic of a zinc finger motif. The finger region maps from amino acids 302 through 320 and has the sequence C-302 L K C-305 I K K E Q P S H Y K Y H-317 E K H-320. Previous genetic analysis has shown that the cysteine and histidine sequences and the contiguous S H Y K Y region in the finger are important for DNA replication in vivo. We show here that representative mutations in either of these elements of the finger prevent the assembly of large T antigen into stable hexamers in vitro. These same mutations have a characteristic effect on the interaction of T antigen with the simian virus 40 core origin of replication. The mutant T antigens bind to the central pentanucleotide domain of the core origin but fail to melt the adjacent inverted repeat domain and to untwist the adenine-thymine domain. These defects would prevent the formation of a replication bubble and the initiation of DNA replication. Finger mutations have lesser effects on the helicase function of T antigen and no observable effect on binding of T antigen to the mouse p53 protein. We propose that the zinc finger region contributes to protein-protein interactions essential for the assembly of stable T-antigen hexamers at the origin of replication and that hexamers are needed for subsequent alterations in the structure of origin DNA. We cannot exclude the possibility that the zinc finger region also makes specific contacts with components of origin DNA.     

Critical spatial requirement within the origin of simian virus 40 DNA replication.

We inserted a single base pair into the center of a 27-base-pair palindrome within the replication origin of simian virus 40. The mutation did not directly alter the symmetry of the palindrome or the protein-binding sequences within the palindrome. DNA binding studies showed that subunits of the simian virus 40 A protein (T antigen) bound to each of the four recognition pentanucleotides in the origin palindrome but did so with reduced affinity in comparison with wild-type origins. The mutant origin cloned in a plasmid DNA failed to replicate in COS cells. Thus, precise spatial interactions among subunits of A protein are necessary for stable origin binding and are crucial for subsequent steps in the initiation of DNA replication. Furthermore, any possible functional interactions of the simian virus 40 A protein with cellular DNA would require a great fidelity of protein binding arrangements to initiate cellular DNA replication.     

Purification of the simian virus 40 (SV40) T antigen DNA-binding domain and characterization of its interactions with the SV40 origin.

To better define protein-DNA interactions at a eukaryotic origin, the domain of simian virus 40 (SV40) large T antigen that specifically interacts with the SV40 origin has been purified and its binding to DNA has been characterized. Evidence is presented that the affinity of the purified T antigen DNA-binding domain for the SV40 origin is comparable to that of the full-length T antigen. Furthermore, stable binding of the T antigen DNA-binding domain to the SV40 origin requires pairs of pentanucleotide recognition sites separated by approximately one turn of a DNA double helix and positioned in a head-to-head orientation. Although two pairs of pentanucleotides are present in the SV40 origin, footprinting and band shift experiments indicate that binding is limited to dimer formation on a single pair of pentanucleotides. Finally, it is demonstrated that the T antigen DNA-binding domain interacts poorly with single-stranded DNA.     

Nonspecific DNA binding activity of simian virus 40 large T antigen: evidence for the cooperation of two regions for full activity.

We generated a series of COOH-terminal truncated simian virus 40 large tumor (T) antigens by using oligonucleotide-directed site-specific mutagenesis. The mutant proteins [T(1-650) to T(1-516)] were expressed in insect cells infected with recombinant baculoviruses. T(1-623) and shorter proteins [T(1-621) to T(1-516)] appeared to be structurally changed in a region between residues 269 and 522, as determined by increased sensitivities to trypsin digestion and by altered reactivities to several monoclonal antibodies. These same mutant proteins bound significantly less nonorigin plasmid DNA (15%) and calf thymus DNA (25%) than longer proteins [T(1-625) to T(1-708)]. However, all mutant T antigens exhibited a nearly wild-type level of viral origin-specific DNA binding and binding to a helicase substrate DNA. This indicated that binding to origin and helicase substrate DNAs is separable from about 85% of nonspecific binding to double-stranded DNA. As an independent confirmation that a region distinct from the origin-binding domain (amino acids 147 to 247) is involved in nonspecific DNA binding, we found that up to 96% of this latter activity was specifically inhibited in wild-type T antigen by several monoclonal antibodies which collectively bind to the region between residues 269 and 522. In order to investigate the relationship between the origin-binding domain and the second region, we performed origin-specific DNA binding assays with increasing amounts of calf thymus DNA as competitor. The results suggest that this second region is not an independent nonspecific DNA binding domain. Rather, it most likely cooperates with the origin-binding domain to give rise to wild-type levels of nonspecific DNA binding. Our results further suggest that most of the nonspecific binding to double-stranded DNA is involved in a function other than direct recognition and binding to the pentanucleotides at the replication origin on simian virus 40 DNA.     

The finger domain of simian virus 40 large T antigen controls DNA-binding specificity.

The specificity and regulation of protein-DNA interactions play a crucial role in all aspects of communication between genotype and phenotype in a cell. The large T antigen of simian virus 40 binds to identical, yet quite differently arranged, pentanucleotide motifs in the simian virus 40 control region, sites I and II. Wild-type T antigen preferentially binds site I. We demonstrate that a bacterial peptide encoding residues 1 to 259 (T260) includes the essential amino acids required for binding to both DNA sites but predominantly binds site II. However, a longer peptide (residues 1 to 369; T370) binds almost exclusively to site I. Thus, the addition of amino acids 260 to 369 to the T260 peptide results in the loss of site II binding. This region includes a single putative metal-binding region, and mutation of T370 at either conserved cysteine of the finger results in equal but inefficient binding to both sites. While no metal binding has been shown to be directly associated with this sequence, these results suggest a novel, perhaps structural, function for such a finger motif, since this domain of T antigen appears to play a crucial role in modulating the DNA-binding behavior of T-antigen peptides.     

Subclasses of simian virus 40 large T antigen: differential binding of two subclasses of T antigen from productively infected cells to viral and cellular DNA

Two major subclasses of simian virus 40 (SV40) large T antigen were separated by zone velocity sedimentation of crude extracts from productively infected cells. These subclasses, which have been shown to differ biologically and biochemically ( Fanning et al., 1981), sedimented at 5-6S and 14-16S. The amount of T antigen in each form was estimated by complement fixation and by immunoprecipitation of T antigen from extracts of cells chronically labeled with [35S]methionine. Each form of T antigen was tested for specific binding to end-labeled restriction fragments of SV40 DNA using an immunoprecipitation assay. The 5-6S and 14-16S forms of T antigen both bound specifically to DNA sequences in the SV40 HindIII C fragment. The sequences required for binding both forms were localized in the same 35-bp region of the origin. However, significant differences in binding activity and affinity for specific and nonspecific DNA were demonstrated. These properties suggest that T antigen subclasses may serve different functions in the lytically infected cell.     

An N-terminal deletion mutant of simian virus 40 (SV40) large T antigen oligomerizes incorrectly on SV40 DNA but retains the ability to bind to DNA polymerase alpha and replicate SV40 DNA in vitro.

A peptide encompassing the N-terminal 82 amino acids of simian virus 40 (SV40) large T antigen was previously shown to bind to the large subunit of DNA polymerase alpha-primase (I. Dornreiter, A. Höss, A. K. Arthur, and E. Fanning, EMBO J. 9:3329-3336, 1990). We report here that a mutant T antigen, T83-708, lacking residues 2 to 82 retained the ability to bind to DNA polymerase alpha-primase, implying that it carries a second binding site for DNA polymerase alpha-primase. The mutant protein also retained ATPase, helicase, and SV40 origin DNA-binding activity. However, its SV40 DNA replication activity in vitro was reduced compared with that of wild-type protein. The reduction in replication activity was accompanied by a lower DNA-binding affinity to SV40 origin sequences and aberrant oligomerization on viral origin DNA. Thus, the first 82 residues of SV40 T antigen are not strictly required for its interaction with DNA polymerase alpha-primase or for DNA replication function but may play a role in correct hexamer assembly and efficient DNA binding at the origin.     

Binding of simian virus 40 a protein to DNA with deletions at the origin of replication.

Previous studies with wild-type simian virus 40 DNA have shown that the sequence 5'-GAGGC-3' directs the binding of A protein (T antigen). The functional origin of replication contains four recognition pentanucleotides each of which is separated by a single base pair and arranged a two pairs of direct repetitions that are inverted relative to each other. Analysis of A protein binding to a series of nonviable mutants progressively deleting these contact sites leads to the following conclusions: (i) stable binding of subunits of A protein to three origin pentanucleotides is not sufficient for the initiation of DNA replication, (ii) the stability of DNA binding depends on interactions between bound protein subunits, and (iii) a single pentanucleotide is sufficient to bind and orient a subunit of A protein.