Quantitative analysis of the binding of simian virus 40 large T antigen to DNA

SV40 large T antigen (T-ag) is a multifunctional protein that successively binds to 5'-GAGGC-3' sequences in the viral origin of replication, melts the origin, unwinds DNA ahead of the replication fork, and interacts with host DNA replication factors to promote replication of the simian virus 40 genome. The transition of T-ag from a sequence-specific binding protein to a nonspecific helicase involves its assembly into a double hexamer whose formation is likely dictated by the propensity of T-ag to oligomerize and its relative affinities for the origin as well as for nonspecific double- and single-stranded DNA. In this study, we used a sensitive assay based on fluorescence anisotropy to measure the affinities of wild-type and mutant forms of the T-ag origin-binding domain (OBD), and of a larger fragment containing the N-terminal domain (N260), for different DNA substrates. We report that the N-terminal domain does not contribute to binding affinity but reduces the propensity of the OBD to self-associate. We found that the OBD binds with different affinities to its four sites in the origin and determined a consensus binding site by systematic mutagenesis of the 5'-GAGGC-3' sequence and of the residue downstream of it, which also contributes to affinity. Interestingly, the OBD also binds to single-stranded DNA with an approximately 10-fold higher affinity than to nonspecific duplex DNA and in a mutually exclusive manner. Finally, we provide evidence that the sequence specificity of full-length T-ag is lower than that of the OBD. These results provide a quantitative basis onto which to anchor our understanding of the interaction of T-ag with the origin and its assembly into a double hexamer.     

Structure of the origin-binding domain of simian virus 40 large T antigen bound to DNA

The large T antigen (T-ag) protein binds to and activates DNA replication from the origin of DNA replication (ori) in simian virus 40 (SV40). Here, we determined the crystal structures of the T-ag origin-binding domain (OBD) in apo form, and bound to either a 17 bp palindrome (sites 1 and 3) or a 23 bp ori DNA palindrome comprising all four GAGGC binding sites for OBD. The T-ag OBDs were shown to interact with the DNA through a loop comprising Ser147-Thr155 (A1 loop), a combination of a DNA-binding helix and loop (His203-Asn210), and Asn227. The A1 loop traveled back-and-forth along the major groove and accounted for most of the sequence-determining contacts with the DNA. Unexpectedly, in both T-ag-DNA structures, the T-ag OBDs bound DNA independently and did not make direct protein-protein contacts. The T-ag OBD was also captured bound to a non-consensus site ATGGC even in the presence of its canonical site GAGGC. Our observations taken together with the known biochemical and structural features of the T-ag-origin interaction suggest a model for origin unwinding.     

Mutational analysis of simian virus 40 large T antigen DNA binding sites.

We have tested the effects of various mutations within SV40 T antigen DNA recognition sites I and II on specific T antigen binding using the DNase footprint technique. In addition, the replication of plasmid DNA templates carrying these T antigen binding site mutations was monitored by Southern analysis of transfected DNA in COS cells. Deletion mapping of site I sequences defined a central core of approximately 18 bp that is both necessary and sufficient for T antigen recognition; this region contains the site I contact nucleotides that were previously mapped using methylation-interference and methylation-protection experiments. A similar deletion analysis delineated sequences that impart specificity of binding to site II. We find that T antigen is capable of specific recognition of site II in the absence of site I sequences, indicating that binding to site II in vitro is not dependent on binding of T antigen at site I. Site II binding was not diminished by small deletion or substitution mutations that perturb the 27-bp palindrome central to binding site II, whereas extensive substitution of site II sequences completely eliminated specific site II binding. Analysis of the replication in COS7 cells of plasmids that contain these mutant origins revealed that sequences both at the late side of binding site I and within the site II palindrome are crucial for viral DNA replication, but are not involved in binding T antigen.     

Interactions required for binding of simian virus 40 T antigen to the viral origin and molecular modeling of initial assembly events

The purified T-antigen origin binding domain binds site specifically to site II, the central region of the simian virus 40 core origin. However, in the context of full-length T antigen, the origin binding domain interacts poorly with DNA molecules containing just site II. Here we investigate the contributions of additional core origin regions, termed the flanking sequences, to origin recognition and the assembly of T-antigen hexamers and double hexamers. Results from these studies indicate that in addition to site-specific binding of the T-antigen origin binding domain to site II, T-antigen assembly requires non-sequence-specific interactions between a basic finger in the helicase domain and particular flanking sequences. Related studies demonstrate that the assembly of individual hexamers is coupled to the distortions in the proximal flanking sequence. In addition, the point in the double-hexamer assembly process that is regulated by phosphorylation of threonine 124, the sole posttranslational modification required for initiation of DNA replication, was further analyzed. Finally, T-antigen structural information is used to model various stages of T-antigen assembly on the core origin and the regulation of this process.     

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.     

Expression of simian virus 40 T antigen in Escherichia coli: localization of T-antigen origin DNA-binding domain to within 129 amino acids.

The genomic coding sequence of the large T antigen of simian virus 40 (SV40) was cloned into an Escherichia coli expression vector by joining new restriction sites, BglII and BamHI, introduced at the intron boundaries of the gene. Full-length large T antigen, as well as deletion and amino acid substitution mutants, were inducibly expressed from the lac promoter of pUC9, albeit with different efficiencies and protein stabilities. Specific interaction with SV40 origin DNA was detected for full-length T antigen and certain mutants. Deletion mutants lacking T-antigen residues 1 to 130 and 260 to 708 retained specific origin-binding activity, demonstrating that the region between residues 131 and 259 must carry the essential binding domain for DNA-binding sites I and II. A sequence between residues 302 and 320 homologous to a metal-binding "finger" motif is therefore not required for origin-specific binding. However, substitution of serine for either of two cysteine residues in this motif caused a dramatic decrease in origin DNA-binding activity. This region, as well as other regions of the full-length protein, may thus be involved in stabilizing the DNA-binding domain and altering its preference for binding to site I or site II DNA.     

The role of surface-exposed lysines in wrapping DNA about the bacterial histone-like protein HU

Several basic proteins, including the ubiquitous HU proteins, serve histone-like functions in prokaryotes. Significant sequence conservation exists between HU homologues; yet binding sites varying from 9 to 37 bp have been reported. TF1, an HU homologue with a 37 bp binding site that is encoded by the Bacillus subtilis bacteriophage SPO1, binds with nM affinity to DNA that contains 5-hydroxymethyluracil (hmU) in place of thymine and to T-containing DNA with loops. We evaluated the contribution of three conserved lysines to specifying the length of the binding site and show that Lys3 is critical for maintaining a long binding site in T-containing DNA: A mutant protein in which Lys3 is replaced with Gln(TF1-K3Q) is completely deficient in forming a stable complex. The affinity for 37 bp hmU-containing DNA is also reduced, from approximately 3 nM for wild-type TF1 to approximately 90 nM for TF1-K3Q. The decrease in affinity of TF1-K3Q for hmU-containing DNA > or = 25 bp suggests that Lys3 contacts DNA 8-9 bp distal to the sites of kinking. We propose that Lys3 forms an internal saltbridge to Asp26 in HU homologues characterized by shorter binding sites and that its surface exposure, and hence a longer binding site, may correlate with absence of this aspartate.     

Polyomavirus Large T Antigen Binds Cooperatively to Its Multiple Binding Sites in the Viral Origin of DNA Replication

Polyomavirus large T antigen binds to multiple 5′-G(A/G)GGC-3′ pentanucleotide sequences in sites 1/2, A, B, and C within and adjacent to the origin of viral DNA replication on the polyomavirus genome. We asked whether the binding of large T antigen to one of these sites could influence binding to other sites. We discovered that binding to origin DNA is substantially stronger at pH 6 to 7 than at pH 7.4 to 7.8, a range often used in DNA binding assays. Large T antigen-DNA complexes formed at pH 6 to 7 were stable, but a fraction of these complexes dissociated at pH 7.6 and above upon dilution or during electrophoresis. Increased binding at low pH is therefore due at least in part to increased stability of protein-DNA complexes, and binding at higher pH values is reversible. Binding to fragments of origin DNA in which one or more sites were deleted or inactivated by point mutations was measured by nitrocellulose filter binding and DNase I footprinting. The results showed that large T antigen binds cooperatively to its four binding sites in viral DNA, suggesting that the binding of this protein to one of these sites stabilizes its binding to other sites via protein-protein contacts. Sites A, B, and C may therefore augment DNA replication by facilitating the binding of large T antigen to site 1/2 at the replication origin. ATP stabilized large T antigen-DNA complexes against dissociation in the presence, but not the absence, of site 1/2, and ATP specifically enhanced protection against DNase I digestion in the central 10 to 12 bp of site 1/2, at which hexamers are believed to form and begin unwinding DNA. We propose that large T antigen molecules bound to these multiple sites on origin DNA interact with each other to form a compact protein-DNA complex and, furthermore, that ATP stimulates their assembly into hexamers at site 1/2 by a “handover” mechanism mediated by these protein-protein contacts.     

Effects of in vitro dephosphorylation on DNA-binding and DNA helicase activities of simian virus 40 large tumor antigen.

Simian virus 40 large T antigen is a phosphoprotein with two clusters of phosphorylation sites. Each cluster includes four serine residues and one threonine residue. In vitro treatment with intestinal alkaline phosphatase removes the phosphate groups from the serine but not from the threonine residues. Potato acid phosphatase additionally dephosphorylates the phosphothreonine (Thr-124) in the N-terminal cluster but does not attack the phosphothreonine in the C-terminal cluster (Thr-701). Two biochemical functions of untreated and partially dephosphorylated T antigen were assayed, namely, its specific DNA-binding property and its DNA helicase activity. After treatment with alkaline phosphatase, T antigen had a severalfold higher affinity for the specific binding sites in the viral genomic control region, in particular, for binding site II in the origin of replication. However, T antigen, when dephosphorylated by acid phosphatase, had DNA-binding properties similar to those of the untreated control. Neither alkaline nor acid dephosphorylation affected the DNA helicase activity of T antigen.     

Simian virus 40 large T antigen untwists DNA at the origin of DNA replication.

Simian virus 40 large tumor antigen (SV40 T antigen) untwists DNA at the SV40 replication origin. In the presence of ATP, T antigen shifted the average linking number of an SV40 origin-containing plasmid topoisomer distribution. The loss of up to two helical turns was detected. The reaction required the presence of the 64-base pair core origin of replication containing T antigen DNA binding site II; binding site I had no effect on the untwisting reaction. The presence of human single-stranded DNA binding protein (SSB) slightly reduced the degree of untwisting in the presence of ATP. ATP hydrolysis was not required since untwisting occurred in the presence of nonhydrolyzable analogs of ATP. However, in the presence of a nonhydrolyzable analog of ATP, the requirement for the SV40 origin sequence was lost. The origin requirement for DNA untwisting was also lost in the absence of dithiothreitol. The origin-specific untwisting activity of T antigen is distinct from its DNA helicase activity, since helicase activity does not require the SV40 origin but does require ATP hydrolysis. The lack of a requirement for SSB or ATP hydrolysis and the reduction in the pitch of the DNA helix by just a few turns at the replication origin distinguishes this reaction from the T antigen-mediated DNA unwinding reaction, which results in the formation of a highly underwound DNA molecule. Untwisting occurred without a lag after the start of the reaction, whereas unwound DNA was first detected after a lag of 10 min. It is proposed that the formation of a multimeric T antigen complex containing untwisted DNA at the SV40 origin is a prerequisite for the initiation of DNA unwinding and replication.     

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.     

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.     

Characterization of the herpes simplex virus origin binding protein interaction with OriS.

The origin binding protein (OBP) of herpes simplex virus (HSV), which is essential for viral DNA replication, binds specifically to sequences within the viral replication origin(s) (for a review, see Challberg, M.D., and Kelly, T. J. (1989) Annu. Rev. Biochem. 58, 671-717). Using either a COOH-terminal OBP protein A fusion or the full-length protein, each expressed in Escherichia coli, we investigated the interaction of OBP with one HSV origin, OriS. Binding of OBP to a set of binding site variant sequences demonstrates that the 10-base pair sequence, 5' CGTTCGCACT 3', comprises the OBP-binding site. This sequence must be presented in the context of at least 15 total base pairs for high affinity binding, Ka = approximately 0.3 nM. Single base pair mutations in the central CGC sequence lower the affinity by several orders of magnitude, whereas a substitution at any of the other seven positions reduces the affinity by 10-fold or less. OBP binds with high affinity to duplex DNA containing mismatched base pairs. This property is exploited to analyze OBP binding to DNA heteroduplexes containing singly substituted mutant and wild-type DNA strands. For positions 2, 3, 5, 6, 7, 8, and 9, substitutions are tolerated on one or the other DNA strand, indicating that base-mediated interactions are limited to one base of each pair. For both Boxes I and II, these interactions are localized to one face of the DNA helix, forming a recognition surface in the major groove. In OriS, the 31 base pairs which separate Boxes I and II orient the two interaction surfaces to the same side of the DNA.     

Asymmetric assembly of Merkel cell polyomavirus large T-antigen origin binding domains at the viral origin

The double-stranded DNA polyomavirus Merkel cell polyomavirus (MCV) causes Merkel cell carcinoma, an aggressive but rare human skin cancer that most often affects immunosuppressed and elderly persons. As in other polyomaviruses, the large T-antigen of MCV recognizes the viral origin of replication by binding repeating G(A/G)GGC pentamers. The spacing, number, orientation, and necessity of repeats for viral replication differ, however, from other family members such as SV40 and murine polyomavirus. We report here the 2.9 Å crystal structure of the MCV large T-antigen origin binding domain (OBD) in complex with a DNA fragment from the MCV origin of replication. Consistent with replication data showing that three of the G(A/G)GGC-like binding sites near the center of the origin are required for replication, the crystal structure contains three copies of the OBD. This stoichiometry was verified using isothermal titration calorimetry. The affinity for G(A/G)GGC-containing double-stranded DNA was found to be ∼ 740 nM, approximately 8-fold weaker than the equivalent domain in SV40 for the analogous region of the SV40 origin. The difference in affinity is partially attributable to DNA-binding residue Lys331 (Arg154 in SV40). In contrast to SV40, a small protein-protein interface is observed between MCV OBDs when bound to the central region of the origin. This protein-protein interface is reminiscent of that seen in bovine papilloma virus E1 protein. Mutational analysis indicates, however, that this interface contributes little to DNA binding energy.     

High-affinity DNA binding of HU protein from the hyperthermophile Thermotoga maritima

Prokaryotic genomes are compacted by association with small basic proteins, generating what has been termed bacterial chromatin. The ubiquitous DNA-binding protein HU serves this function. DNA-binding properties of HU from the hyperthermophilic eubacterium Thermotoga maritima are shown here to differ significantly from those characteristic of previously described HU homologs. Electrophoretic mobility shift analyses show that T. maritima HU (TmHU) binds double-stranded DNA with high affinity (K(d)=5.6(+/-0.7) nM for 37 bp DNA). Equivalent affinity is observed between 4 degrees C and 45 degrees C. TmHU has higher affinity for DNA containing a set of 4 nt loops separated by 9 bp (K(d)=1.4(+/-0.3) nM), consistent with its introduction of two DNA kinks. Using DNA probes of varying length, the optimal binding site for TmHU is estimated at 37 bp, in sharp contrast to the 9-10 bp binding site reported for other HU homologs. Alignment of >60 HU sequences demonstrates significant sequence conservation: A DNA-intercalating proline residue is almost universally conserved, and it is preceded by arginine and asparagine in most sequences, generating a highly conserved RNP motif; V substitutes for R only in HU from Thermotoga, Thermus and Deinococcus. A fivefold increase in DNA-binding affinity is observed for TmHU in which V is replaced with R (TmHU-V61R; K(d)=1.1(+/-0.2) nM), but a change in the trajectory of DNA flanking the sites of DNA intercalation is inferred from analysis of TmHU-V61R binding to DNA modified with 4 nt loops or with substitutions of 5-hydroxymethyluracil for thymine. Survival in extreme environments places unique demands on protection of genomic DNA from thermal destabilization and on access of DNA to the cellular machinery, demands that may be fulfilled by the specific DNA-binding properties of HU and by the fine structure of the bacterial chromatin.     

ATP enhances the binding of simian virus 40 large T antigen to the origin of replication.

Simian virus 40 large T antigen initiates DNA replication by binding to the origin of replication. We examined the binding of T antigen to origin regions I, II, and III under conditions designed for efficient in vitro replication functions. We found that 4 mM ATP enhanced the binding of T antigen to regions I and II of the origin DNA by 4- to 20-fold. DNase-footprinting and fragment assays showed that ATP extended the DNase protection domain of T antigen bound to region II by 5 to 10 base pairs at both ends of the core origin of replication. This alteration suggests a change in the conformation of T antigen, bound DNA, or both.     

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

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.