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.     

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.     

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.     

Origin binding by a 100,000-dalton super-T antigen from SVT2 cells.

The SVT2 line of simian virus 40-transformed mouse cells expresses little or no wild-type-size A protein (T antigen). Instead, a variant form is produced in these cells that is larger than normal-size A protein. This variant form has an Mr of 100,000 (100K super-T antigen) and is found primarily in complexes with the host-cell-coded p53 protein. Binding of the 100K super-T antigen to simian virus 40 origin region DNA was assayed by immunoprecipitation of super-T antigen-DNA complexes and then digestion with DNase I. DNA sequences associated with super-T antigen were protected from digestion and retained in the immune complex, while unprotected sequences were digested and released. The 100K super-T antigen efficiently protects DNA sequences in the previously defined regions I and II (P. Tegtmeyer, B. A. Lewton, A. L. DeLucia, V. G. Wilson, and K. Ryder, J. Virol. 46:151-161, 1983). Within region II (the origin of replication), the pattern and size of protected fragments are identical for super-T antigen and purified wild-type A protein. Thus, even though super-T antigen is larger than wild-type A protein, both must bind with the same alignment on origin DNA. Furthermore, complexes between the host-cell-coded p53 protein and the 100K super-T antigen also retain the ability to bind in regions I and II.     

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.     

Role of the hydrophilic channels of simian virus 40 T-antigen helicase in DNA replication

The simian virus 40 (SV40) hexameric helicase consists of a central channel and six hydrophilic channels located between adjacent large tier domains within each hexamer. To study the function of the hydrophilic channels in SV40 DNA replication, a series of single-point substitutions were introduced at sites not directly involved in protein-protein contacts. The mutants were characterized biochemically in various ways. All mutants oligomerized normally in the absence of DNA. Interestingly, 8 of the 10 mutants failed to unwind an origin-containing DNA fragment and nine of them were totally unable to support SV40 DNA replication in vitro. The mutants fell into four classes based on their biochemical properties. Class A mutants bound DNA normally and had normal ATPase and helicase activities but failed to unwind origin DNA and support SV40 DNA replication. Class B mutants were compromised in single-stranded DNA and origin DNA binding at low protein concentrations. They were defective in helicase activity and unwinding of the origin and in supporting DNA replication. Class C and D mutants possessed higher-than-normal single-stranded DNA binding activity at low protein concentrations. The class C mutants failed to separate origin DNA and support DNA replication. The class D mutants unwound origin DNA normally but were compromised in their ability to support DNA replication. Taken together, these results suggest that the hydrophilic channels have an active role in the unwinding of SV40 DNA from the origin and the placement of the resulting single strands within the helicase.     

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.     

Protein-induced bending of the simian virus 40 origin of replication

A 3.5 S protein, isolated from mammalian nuclei, specifically binds to DNA fragments containing the simian virus 40 (SV40) origin of replication. Two distinct nucleoprotein complexes are formed, a complex with high electrophoretic mobility carrying probably only one protein molecule, and a complex with reduced electrophoretic mobility carrying probably two protein molecules per DNA fragment. Band shift competition as well as methylation interference assays locate the binding site of the protein in the A + T-rich "late" region of the origin between SV40 nucleotides 13 and 35. The late origin binding (LOB) protein and T antigen bind simultaneously to adjacent sites in the origin. Using circularly permuted DNA fragments of identical lengths we show that the LOB protein induces pronounced bending of the origin fragment. The bending center maps at the 5' end of the adenine tract with one bound protein molecule and at the 3' end when two LOB proteins are bound to one origin fragment.     

Two conditional tsA mutant simian virus 40 T antigens display marked differences in thermal inactivation.

We have characterized the simian virus 40 (SV40) origin-containing DNA (ori-DNA) replication functions of two SV40 conditional mutant T antigens: tsA438 A-V (tsA58) and tsA357 R-K (tsA30). Both tsA mutant T antigens, immunopurified from recombinant baculovirus-infected insect cells, mediated replication of SV40 ori-DNA in vitro to similar extents as did wild-type T antigen in reactions at 33 degrees C. However, at 41 degrees C, the restrictive temperature, while tsA438 T antigen still generated substantial levels of replication products, tsA357 T antigen did not support any detectable DNA synthesis. Furthermore, preincubation for approximately fourfold-longer time periods at 41 degrees C was required to heat inactivate tsA438 T antigen than to heat inactivate tsA357 T antigen. Unexpectedly, results of analyses of the various DNA replication activities of the two mutant T antigens did not correlate with results from ori-DNA replication reactions. In particular, although tsA357 T antigen was incapable of mediating replication at 41 degrees C at all protein concentrations examined, it displayed either wild-type levels or only partial reductions of the several T-antigen replication-associated activities. These data suggest either that tsA357 T antigen is defective in an as yet unidentified replication function of T antigen or that the combination of its partial defects result in a protein that is unable to support replication. The data also show that two conditional mutant T antigens can be markedly different with respect to thermal sensitivity.     

Patterns of strongly protein-associated simian virus 40 DNA replication intermediates resulting from exposures to specific topoisomerase poisons

Exposure of infected CV-1 cells to specific type I and type II topoisomerase poisons caused strong protein association with distinct subsets of simian virus 40 (SV40) DNA replication intermediates. On the basis of the known specificity and mechanisms of action of these drugs, the proteins involved are assumed to be the respective topoisomerases. Camptothecin, a topoisomerase I poison, caused strong protein association with form II (relaxed circular) and form III (linear) viral genomes and replication intermediates having broken DNA replication forks but not with form I (superhelical) viral DNA or normal late replication intermediates which were present. In contrast, type II topoisomerase poisons caused completely replicated forms and late viral replication forms to be tightly bound to protein--some to a greater extent than others. Different type II topoisomerase inhibitors caused distinctive patterns of protein association with the replication intermediates present. Both intercalating and nonintercalating type II topoisomerase poisons caused a small amount of form I (superhelical) SV40 DNA to be protein-associated in vivo. The protein complex with form I viral DNA was entirely drug-dependent and strong, but apparently noncovalent. The protein associated with form I DNA may represent a drug-stabilized "topological complex" between type II topoisomerase and SV40 DNA.     

Association of simian virus 40 T antigen with replicating nucleoprotein complexes of simian virus 40.

An immunoprecipitation assay was established for simian virus 40 T-antigen-bound nucleoprotein complexes by means of precipitation with sera from hamsters bearing simian virus 40-induced tumors. About 80% of simian virus 40 replicating nucleoprotein complexes in various stages of replication were immunoprecipitated. In contrast, less than 21% of mature nucleoprotein complexes were immunoprecipitated. Pulse-chase experiments showed that T antigen was lost from most of the nucleoprotein complexes concurrently with completion of DNA replication. T antigen induced by dl-940, a mutant with a deletion in the region coding for small T antigen, was also associated with most of the replicating nucleoprotein complexes. Once bound with replicating nucleoprotein complexes at the permissive temperature, thermolabile T antigen induced by tsA900 remained associated with the complexes during elongation of the replicating DNA chain at the restrictive temperature. These results suggest that simian virus 40 T antigen (probably large T antigen) associates with nucleoprotein complexes at or before initiation of DNA replication and that the majority of the T antigen dissociates from the nucleoprotein complexes simultaneously with completion of DNA replication.     

Fine structure of polyoma virus DNA.

A fine structure map of polyoma DNA has been made based on cleavage with a number of restriction endonucleases (including HaeII and III, BamI, HindII and III, BumI, HpaII, and in part, HphI) and depurination of wild-type DNA, the eight HpaII restriction fragments and some HaeIII fragments. This analysis has made possible some correlation with simian virus 40 DNA, and has facilitated detailed examination of various polyoma strains and variants. Sequences from the region of the origin of DNA replication have been examined.     

Specific interaction between the initiator protein (Rep) and origin of plasmid ColE2-P9

The replication initiator protein (Rep) of plasmid ColE2-P9 (ColE2) is multifunctional. We are interested in how Rep binds to the origin (Ori) to perform various functions. We used the wild type and variants of Rep to study the Rep-Ori interaction by both in vitro and in vivo approaches, including biochemical analyses of protein-DNA interactions and an in vivo replication assay. We identified three regions (I, II, and III) of Rep, located in the C-terminal half, and three corresponding binding sites (I, II, and III) in Ori which are important for Rep-Ori interaction. We showed that region I, containing a putative helix-turn-helix motif, is necessary and sufficient for specific Ori recognition, interacting with site I of the origin DNA from the major groove. Region II interacts with site II of the origin DNA, from the adjacent minor groove in the left half of Ori, and region III interacts with site III, next to the template sequence for primer synthesis, which is one and one-half turn apart from site I on the opposite surface of the origin DNA. A putative linker region located between the two DNA binding domains (regions II and III) was identified, which might provide Rep an extended conformation suitable for binding to the two separate sites in Ori. Based on the results presented in this paper, we propose a model for Rep-Ori interaction in which Rep binds to Ori as a monomer.     

Thermally inactivated simian virus 40 tsA58 mutant T antigen cannot initiate viral DNA replication in vitro.

The mutation in the temperature-sensitive tsA58 mutant T antigen (Ala-438----Val) lies within the presumptive ATP-binding fold. We have constructed a recombinant baculovirus that expresses large quantities of the tsA58 T antigen in infected insect cells. The mutant T antigen mediated simian virus 40 origin-containing DNA (ori-DNA) synthesis in vitro to nearly the same extent as similar quantities of wild-type T antigen at 33 degrees C. However, if wild-type and tsA58 T antigens were heated at 41 degrees C in replication extracts prior to addition of template DNA, the tsA58 T antigen but not the wild type was completely inactivated. The mutant protein displayed greater thermosensitivity for many of the DNA replication activities of T antigen than did the wild-type protein. Some of the replication functions of tsA58 T antigen were differentially affected depending on the presence or absence of ATP during the preheating period. When tsA58 T antigen was preheated in the presence of ATP at 41 degrees C for a time sufficient to completely inactivate its ability to replicate ori-DNA in vitro, it displayed substantial ATPase and normal DNA helicase activities. Conversely, when preheated in the absence of nucleotide, it completely lost both ATPase and helicase activities. Preheating tsA58 T antigen, even in the presence of ATP, led to drastic reductions in its ability to bind to and unwind DNA containing the replication origin. The mutant T antigen also displayed thermosensitivity for binding to and unwinding nonspecific double-stranded DNA in the presence of ATP. Our results suggest that the interactions of T antigen with ATP that are involved in T-antigen DNA binding and DNA helicase activities are different. Moreover, we conclude, consistent with its phenotype in vivo, that the tsA58 T antigen is defective in the initiation but not in the putative elongation functions of T antigen in vitro.     

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.