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.     

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.     

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.     

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.     

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.     

Assembly of T-Antigen Double Hexamers on the Simian Virus 40 Core Origin Requires Only a Subset of the Available Binding Sites

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.     

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

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.     

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.     

Methylation of specific cytosine residues enhances simian virus 40 T-antigen binding to origin region DNA.

Specific binding of simian virus 40 large T antigen to origin region DNA requires the interaction of T antigen with multiples of a consensus recognition pentanucleotide sequence (5'-G[T]-A[G]-G-G-C-3'). To assess the interaction of T antigen with cytosine residues in the recognition sequences, bacterial methylases were used to methylate simian virus 40 form I DNA in vitro at specific cytosine residues. Methylation of a subset of the cytosine residues in the pentanucleotide sequences resulted in enhanced binding of T antigen to origin region DNA. Enhanced binding to the methylated pentanucleotides indicates that the methyl groups introduced on this subset of pentanucleotide cytosine residues could not have sterically interfered with the interaction of T antigen with the recognition sequences. This lack of steric interference suggests that T antigen does not make close contact in the major groove with these particular cytosine residues during normal binding.     

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.     

Localized melting and structural changes in the SV40 origin of replication induced by T-antigen.

Replication of simian virus 40 (SV40) DNA is dependent upon the binding of the viral T-antigen to the SV40 origin of replication. Structural changes in the origin of replication induced by binding of T-antigen were probed by chemical modifications of the DNA. In the presence of ATP, T-antigen rendered two of three domains in the SV40 core origin hypersensitive to attack by either dimethyl sulfate or potassium permanganate (KMnO4). One of these domains, the early palindrome, was shown to contain an 8-bp region of melted DNA as determined from methylation of cytosine residues and by nuclease S1 cleavage of methylated DNA. DNA melting was not dependent upon either the hydrolysis of ATP or the binding of T-antigen to an adjacent site (site I). A second domain, the A/T element, was extensively modified by KMnO4 but no significant melting was detected. Rather, the pattern of modification indicates that T-antigen caused a conformational change of the double-stranded DNA in this region. These results suggest that T-antigen, in the presence of ATP, destabilizes the SV40 origin by melting and structurally deforming two flanking regions within the core origin sequence. These DNA structural changes may provide access to other replication factors, allowing complete denaturation of the SV40 origin and the initiation of SV40 DNA replication.     

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.     

Spacing is crucial for coordination of domain functions within the simian virus 40 core origin of replication.

The simian virus 40 core origin of replication is composed of distinct domains that are bracketed by DNA spacers. We created a matched set of insertion mutations in spacer sites to study the spatial relationships among origin domains. Insertions larger than a single base pair severely inhibit replication regardless of the helical phasing between domains. Replication-defective mutations reduce T-antigen binding and T-antigen-induced KMnO4 modifications of DNA to various extents. Mutations in the early half of the origin reduce T-antigen functions in the entire origin, whereas mutations in the late half reduce functions only in that half. Surprisingly, some mutations that severely inhibit DNA replication reduce T-antigen-induced melting and other structural changes within origin DNA to only a limited extent. In contrast, all replication-defective origin mutations prevent T antigen from extending the primary replication bubble beyond the limits of the core origin of replication. We conclude, therefore, that T-antigen-induced events within the core origin must be spatially coordinated for conversion of T-antigen hexamers bound to the core origin into mobile helicase units.     

The crystal structure of the SV40 T-antigen origin binding domain in complex with DNA

DNA replication is initiated upon binding of “initiators” to origins of replication. In simian virus 40 (SV40), the core origin contains four pentanucleotide binding sites organized as pairs of inverted repeats. Here we describe the crystal structures of the origin binding domain (obd) of the SV40 large T-antigen (T-ag) both with and without a subfragment of origin-containing DNA. In the co-structure, two T-ag obds are oriented in a head-to-head fashion on the same face of the DNA, and each T-ag obd engages the major groove. Although the obds are very close to each other when bound to this DNA target, they do not contact one another. These data provide a high-resolution structural model that explains site-specific binding to the origin and suggests how these interactions help direct the oligomerization events that culminate in assembly of the helicase-active dodecameric complex of T-ag.     

Sequence-specific functions of the early palindrome domain within the SV40 core origin of replication.

The early palindrome domain within the SV40 core origin of replication is essential for the initiation of replication. Studies with single point mutants in this region suggested that the early palindrome domain does not function as a cruciform structure, but may be involved in the initiation of SV40 DNA replication in a sequence-specific manner. Two mutants, base-substituted at a primase initiation site nucleotide 5214, showed dramatic decreases in DNA replication in monkey cells. Despite earlier reports to the contrary, disruption of the cruciform configuration or polypyrimidine tract does not invariably lead to lack of replication function, as some mutants unable to form this structure replicate normally. Gel retention assays and DNase I footprinting with the nuclear proteins of monkey cells showed that the 5'GAGGC3' pentanucleotide repeats on either side of early palindrome domain interact with monkey nuclear protein. The early palindrome domain may affect the interaction of SV40 DNA with nuclear protein, and participate in SV40 DNA replication.     

An altered DNA conformation in origin region I is a determinant for the binding of SV40 large T antigen

Seventeen base pairs of DNA from SV40 origin region I encode a tripartite binding site for a dimeric mass of SV40 large T antigen. Two binding components are the directly repeated pentanucleotide sequences 5'-GAGGC-3'/5'-GCCTC-3'. The third component is the asymmetric sequence 5'-TTTTTTG-3'/5'-CAAAAAA-3' that separates the pentanucleotides. Nucleotide-specific features of this spacer element stabilize binding to the adjacent pentanucleotides. We report here that the spacer sequence determines a DNA conformation that correlates with high affinity binding of T antigen. The nature of the spacer sequence suggests that the DNA is bent. We propose that binding of T antigen to region I proceeds through monomer-pentanucleotide interactions and either protein-protein or protein-spacer interactions directed by the spacer-encoded structure.     

E1 recognition sequences in the bovine papillomavirus type 1 origin of DNA replication: interaction between half sites of the inverted repeats.

The E1 protein encoded by bovine papillomavirus type 1 (BPV-1) is required for viral DNA replication, and it binds site specifically to an A/T-rich palindromic sequence within the viral origin of replication. The protein is targeted to this site through cooperative interactions and binding with the virus-encoded E2 protein. To explore the nature of the E1 binding site, we inserted a series of homologous DNA linkers at the center of dyad symmetry within the E1 recognition palindrome. The effects of these modifications indicated that the E1 recognition palindrome can be separated into functional half sites. The series of insertions manifest a phasing relationship with respect to the wild-type BPV-1 genome in that greater biological activity was measured when full integral turns of the DNA helix separated the palindrome than when the separations were half-turns. This phasing pattern of activity was observed to occur in a variety of biological phenotypes, including transformation efficiency, stable plasmid copy number in cell lines established from pooled foci, and transient replication of full-length viral genomes. For replication reporter constructs where E1 and E2 are supplied in trans by the respective expression vectors, distance between the half sites seems to play a major role, yet the phasing relationships are measurable. DNase I protection studies showed that E1 bound very poorly to the construct containing a 5-bp linker, and binding was close to the wild-type level for the 10-bp insertion, consistent with a requirement for a phasing function between half sites with a modulus of 10 bp. Binding to the 15- and 20-bp insertion mutants was weak, but only for the 20-bp insertions was protection over both halves of the palindrome measurable. As it had been previously reported that the 18-bp palindrome contains sufficient nucleotide sequence information for E1 binding, we speculate that a minimal E1 recognition motif is presented in each half site. A comparison between this sequence and that of an upstream region that also binds E1 (the E2RE1 region) revealed a common pentanucleotide motif of APyAAPy. Mutants with substitutions of the ATAAT elements within E2RE1 failed to bind E1 protein. We present models for how repeats of the pentanucleotide sequence may coordinate E1 binding at the dyad symmetry axis of the origin and compare the DNA sequence organization of BPV-1 with those of the simian virus 40 and polyomaviruses at their origins of DNA replication.     

Domain structure of the simian virus 40 core origin of replication.

The simian virus 40 core origin of replication consists of nucleotides 5211 through 31. These 64 base pairs contain three functional domains with strict sequence requirements and two spacer regions with relaxed sequence specificity but precise positional constraints. The early domain extends for 10 contiguous base pairs between nucleotides 5211 and 5220. A 9-base pair spacer from sequences 5221 through 5229 separates the early domain from the 23-base pair central palindrome that directs the binding of T antigen. The late end of the core between nucleotides 12 and 31 also contains spacer and sequence-specific functions that are not yet completely mapped. We propose that the sequence-specific domains are interaction sites for viral and cellular proteins, determinants of DNA conformation, or both. The spacers would position these signals at required distances and rotations relative to one another.