Study of SV40 large T antigen nucleotide specificity for DNA unwinding.

BACKGROUND: Simian Virus 40 (SV40) Large Tumor Antigen (LT) is an essential enzyme that plays a vital role in viral DNA replication in mammalian cells. As a replicative helicase and initiator, LT assembles as a double-hexamer at the SV40 origin to initiate genomic replication. In this process, LT converts the chemical energy from ATP binding and hydrolysis into the mechanical work required for unwinding replication forks. It has been demonstrated that even though LT primarily utilizes ATP to unwind DNA, other NTPs can also support low DNA helicase activity. Despite previous studies on specific LT residues involved in ATP hydrolysis, no systematic study has been done to elucidate the residues participating in the selective usage of different nucleotides by LT. In this study, we performed a systematic mutational analysis around the nucleotide pocket and identified residues regulating the specificity for ATP, TTP and UTP in LT DNA unwinding. METHODS: We performed site-directed mutagenesis to generate 16 LT nucleotide pocket mutants and characterized each mutant's ability to unwind double-stranded DNA, oligomerize, and bind different nucleotides using helicase assays, size-exclusion chromatography, and isothermal titration calorimetry, respectively. RESULTS: We identified four residues in the nucleotide pocket of LT, cS430, tK419, cW393 and cL557 that selectively displayed more profound impact on using certain nucleotides for LT DNA helicase activity. CONCLUSION: Little is known regarding the mechanisms of nucleotide specificity in SV40 LT DNA unwinding despite the abundance of information available for understanding LT nucleotide hydrolysis. The systematic residue analysis performed in this report provides significant insight into the selective usage of different nucleotides in LT helicase activity, increasing our understanding of how LT may structurally prefer different energy sources for its various targeted cellular activities.

The structure of SV40 large T hexameric helicase in complex with AT-rich origin DNA.

DNA replication is a fundamental biological process. The initial step in eukaryotic DNA replication is the assembly of the pre-initiation complex, including the formation of two head-to-head hexameric helicases around the replication origin. How these hexameric helicases interact with their origin dsDNA remains unknown. Here, we report the co-crystal structure of the SV40 Large-T Antigen (LT) hexameric helicase bound to its origin dsDNA. The structure shows that the six subunits form a near-planar ring that interacts with the origin, so that each subunit makes unique contacts with the DNA. The origin dsDNA inside the narrower AAA+ domain channel shows partial melting due to the compression of the two phosphate backbones, forcing Watson-Crick base-pairs within the duplex to flip outward. This structure provides the first snapshot of a hexameric helicase binding to origin dsDNA, and suggests a possible mechanism of origin melting by LT during SV40 replication in eukaryotic cells.

High-throughput, high-force probing of DNA-protein interactions with magnetic tweezers.

Recent advances in high-throughput single-molecule magnetic tweezers have paved the way for obtaining information on individual molecules as well as ensemble-averaged behavior in a single assay. Here we describe how to design robust high-throughput magnetic tweezers assays that specifically require application of high forces (>20pN) for prolonged periods of time (>1000s). We elaborate on the strengths and limitations of the typical construct types that can be used and provide a step-by-step guide towards a high tether yield assay based on two examples. Firstly, we discuss a DNA hairpin assay where force-induced strand separation triggers a tight interaction between DNA-binding protein Tus and its binding site Ter, where forces up to 90pN for hundreds of seconds were required to dissociate Tus from Ter. Secondly, we show how the LTag helicase of Simian virus 40 unwinds dsDNA, where a load of 36pN optimizes the assay readout. The approaches detailed here provide guidelines for the high-throughput, quantitative study of a wide range of DNA-protein interactions.

The conserved core enzymatic activities and the distinct dynamics of polyomavirus large T antigens.

Several human polyomaviruses including JCV, BKV and TSV are associated with diseases, particularly in immunosuppressed patients. While the large T antigen (LT) encoded by the monkey polyomavirus SV40 is well studied, and possesses intrinsic ATPase and DNA helicase activities, the LTs of the human polyomaviruses are relatively uncharacterized. In order to evaluate whether these enzymatic activities, which are required for viral DNA replication, are conserved between polyomaviruses, we performed a comparative study using the LTs from JCV, TSV and SV40. The ATPase and DNA helicase activities and the interaction with the cellular tumor suppressor p53 were assayed for the purified Zn-ATPase domains of the three LTs. We found that all Zn-ATPases were active ATPases. The Zn-ATPase domains also functioned as DNA helicases, although the measured kinetic constants differed among the three proteins. In addition, when tested against four small molecule ATPase inhibitors, the Zn-ATPase domains of TSV was more resistant than that of SV40 and JCV. Our results show that, while LTs from JCV and TSV share the core ATPase and DNA helicase activities, they possess important functional differences that might translate into their respective abilities to infect and replicate in hosts.

Use of the SYBR Green dye for measuring helicase activity.

Here we describe a non-radioactive assay that exploits the fluorescent dye SYBR Green to measure the helicase enzyme activity. SYBR Green I emits fluorescence upon intercalation with double-stranded DNA or RNA. The fluorescence is lost proportionally as the nucleic acid is converted to single strands by a helicase, and this decrease in fluorescence intensity can be used to measure the activity of the helicase enzyme. The reaction was prepared by mixing a double-stranded substrate with the helicase enzyme, buffer, ATP and SYBR Green I. After completion, the reaction was terminated by EDTA and fluorescence was measured. Using this technique, a linear increase in substrate release was observed with increasing time and helicase concentrations. The assay described here is speedy, efficient and economical; it holds promise for use in large-scale screening of drugs that target helicases.

Bypass of a protein barrier by a replicative DNA helicase.

Replicative DNA helicases generally unwind DNA as a single hexamer that encircles and translocates along one strand of the duplex while excluding the complementary strand (known as steric exclusion). By contrast, large T antigen, the replicative DNA helicase of the simian virus 40 (SV40), is reported to function as a pair of stacked hexamers that pumps double-stranded DNA through its central channel while laterally extruding single-stranded DNA. Here we use single-molecule and ensemble assays to show that large T antigen assembled on the SV40 origin unwinds DNA efficiently as a single hexamer that translocates on single-stranded DNA in the 3'-to-5' direction. Unexpectedly, large T antigen unwinds DNA past a DNA-protein crosslink on the translocation strand, suggesting that the large T antigen ring can open to bypass bulky adducts. Together, our data underscore the profound conservation among replicative helicase mechanisms, and reveal a new level of plasticity in the interactions of replicative helicases with DNA damage.

A screen for modulators of large T antigen's ATPase activity uncovers novel inhibitors of Simian Virus 40 and BK virus replication.

New polyomaviruses are continually being identified, and it is likely that links between this virus family and disease will continue to emerge. Unfortunately, a specific treatment for polyomavirus-associated disease is lacking. Because polyomaviruses express large Tumor Antigen, TAg, we hypothesized that small molecule inhibitors of the essential ATPase activity of TAg would inhibit viral replication. Using a new screening platform, we identified inhibitors of TAg's ATPase activity. Lead compounds were moved into a secondary assay, and ultimately two FDA approved compounds, bithionol and hexachlorophene, were identified as the most potent TAg inhibitors known to date. Both compounds inhibited Simian Virus 40 replication as assessed by plaque assay and quantitative PCR. Moreover, these compounds inhibited BK virus, which causes BKV Associated Nephropathy. In neither case was host cell viability compromised at these concentrations. Our data indicate that directed screening for TAg inhibitors is a viable method to identify polyomavirus inhibitors, and that bithionol and hexachlorophene represent lead compounds that may be further modified and/or ultimately used to combat diseases associated with polyomavirus infection.

Hexameric helicase deconstructed: interplay of conformational changes and substrate coupling.

Hexameric helicases are molecular motor proteins that utilize energy obtained from ATP hydrolysis to translocate along and/or unwind nucleic acids. In this study, we investigate the dynamic behavior of the Simian Virus 40 hexameric helicase bound to DNA by performing molecular dynamics simulations employing a coarse-grained model. Our results elucidate the two most important molecular features of the helicase motion. First, the attractive interactions between the DNA-binding domain of the helicase and the DNA backbone are essential for the helicase to exhibit a unidirectional motion along the DNA strand. Second, the sequence of ATP binding at multiple binding pockets affects the helicase motion. Specifically, concerted ATP binding does not generate a unidirectional motion of the helicase. It is only when the binding of ATP occurs sequentially from one pocket to the next that the helicase moves unidirectionally along the DNA. Interestingly, in the reverse order of sequential ATP binding, the helicase also moves unidirectionally but in the opposite direction. These observations suggest that in nature ATP molecules must distinguish between different available ATP binding pockets of the hexameric helicase in order to function efficiently. To this end, simulations reveal that the binding of ATP in one pocket induces an opening of the next ATP-binding pocket and such an asymmetric deformation may coordinate the sequential ATP binding in a unidirectional manner. Overall, these findings may provide clues toward understanding the mechanism of substrate translocation in other motor proteins.

Structure of a DNA polymerase alpha-primase domain that docks on the SV40 helicase and activates the viral primosome.

DNA polymerase alpha-primase (pol-prim) plays a central role in DNA replication in higher eukaryotes, initiating synthesis on both leading and lagging strand single-stranded DNA templates. Pol-prim consists of a primase heterodimer that synthesizes RNA primers, a DNA polymerase that extends them, and a fourth subunit, p68 (also termed B-subunit), that is thought to regulate the complex. Although significant knowledge about single-subunit primases of prokaryotes has accumulated, the functions and regulation of pol-prim remain poorly understood. In the SV40 replication model, the p68 subunit is required for primosome activity and binds directly to the hexameric viral helicase T antigen, suggesting a functional link between T antigen-p68 interaction and primosome activity. To explore this link, we first mapped the interacting regions of the two proteins and discovered a previously unrecognized N-terminal globular domain of p68 (p68N) that physically interacts with the T antigen helicase domain. NMR spectroscopy was used to determine the solution structure of p68N and map its interface with the T antigen helicase domain. Structure-guided mutagenesis of p68 residues in the interface diminished T antigen-p68 interaction, confirming the interaction site. SV40 primosome activity of corresponding pol-prim mutants decreased in proportion to the reduction in p68N-T antigen affinity, confirming that p68-T antigen interaction is vital for primosome function. A model is presented for how this interaction regulates SV40 primosome activity, and the implications of our findings are discussed in regard to the molecular mechanisms of eukaryotic DNA replication initiation.

A computational analysis of ATP binding of SV40 large tumor antigen helicase motor.

Simian virus 40 large tumor antigen (LTag) is an efficient helicase motor that unwinds and translocates DNA. The DNA unwinding and translocation of LTag is powered by ATP binding and hydrolysis at the nucleotide pocket between two adjacent subunits of an LTag hexamer. Based on the set of high-resolution hexameric structures of LTag helicase in different nucleotide binding states, we simulated a conformational transition pathway of the ATP binding process using the targeted molecular dynamics method and calculated the corresponding energy profile using the linear response approximation (LRA) version of the semi-macroscopic Protein Dipoles Langevin Dipoles method (PDLD/S). The simulation results suggest a three-step process for the ATP binding from the initial interaction to the final tight binding at the nucleotide pocket, in which ATP is eventually "locked" by three pairs of charge-charge interactions across the pocket. Such a "cross-locking" ATP binding process is similar to the binding zipper model reported for the F1-ATPase hexameric motor. The simulation also shows a transition mechanism of Mg2+ coordination to form the Mg-ATP complex during ATP binding, which is accompanied by the large conformational changes of LTag. This simulation study of the ATP binding process to an LTag and the accompanying conformational changes in the context of a hexamer leads to a refined cooperative iris model that has been proposed previously.

Real-time investigation of SV40 large T-antigen helicase activity using surface plasmon resonance.

The simian virus 40 (SV40) genome is a model system frequently employed for investigating eukaryotic replication. Large T-antigen (T-ag) is a viral protein responsible for unwinding the SV40 genome and recruiting necessary host factors prior to replication. In addition to duplex unwinding T-ag possesses G-quadruplex DNA helicase activity, the physiological consequence of which is unclear. However, formation of G-quadruplex DNA structures may be involved in genome maintenance and function, and helicase activity to resolve these structures may be necessary for efficient replication. We report the first real-time investigation of SV40 T-ag helicase activity using surface plasmon resonance (SPR). In the presence of ATP, T-ag was observed to bind to immobilized single-stranded DNA, forked duplex DNA, and the human telomeric foldover quadruplex DNA sequence. Inhibition of T-ag duplex helicase activity was observable in real-time and the intramolecular quadruplex was unwound.

Inhibition of Simian virus 40 large T antigen helicase activity by fluoroquinolones.

BACKGROUND: Fluoroquinolones represent a potent group of antibiotics that inhibit bacterial DNA replication by targeting the essential bacterial enzymes gyrase and topoisomerase IV. Inhibition of gyrase activity by quinolones involves the interaction of these drugs with the helicase component of bacterial gyrase. DNA tumour viruses also encode helicases that are essential for their DNA replication in the host. METHODS: In this study we have evaluated the effect of fluoroquinolones on viral DNA replication using the DNA tumour virus Simian virus 40 (SV40) as our model. Four different fluoroquinolones, namely, levofloxacin, trovafloxacin, ciprofloxacin and ofloxacin, were tested for their ability to inhibit viral DNA replication. RESULTS: We show here that all four quinolones tested were effective in the inhibition of SV40 plaque formation and DNA replication in CV1-P cells. In addition, we found that each of these quinolones was inhibitory to the helicase activity of SV40 large tumour antigen. CONCLUSIONS: Fluoroquinolones and their derivates may therefore be useful in the treatment and/or prevention of infection by SV40-homologous human DNA viruses that encode helicase activity for their survival.

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.

Crystal structure of SV40 large T-antigen bound to p53: interplay between a viral oncoprotein and a cellular tumor suppressor.

The transformation potential of Simian Virus 40 depends on the activities of large T-antigen (LTag), which interacts with several cellular tumor suppressors including the important "guardian" of the genome, p53. Inhibition of p53 function by LTag is necessary for both efficient viral replication and cellular transformation. We determined the crystal structure of LTag in complex with p53. The structure reveals an unexpected hexameric complex of LTag binding six p53 monomers. Structure-guided mutagenesis of LTag and p53 residues supported the p53-LTag interface defined by the complex structure. The structure also shows that LTag binding induces dramatic conformational changes at the DNA-binding area of p53, which is achieved partially through an unusual "methionine switch" within p53. In the complex structure, LTag occupies the whole p53 DNA-binding surface and likely interferes with formation of a functional p53 tetramer. In addition, we showed that p53 inhibited LTag helicase function through direct complex formation.

Contrahelicase activity of the mitochondrial transcription termination factor mtDBP.

The sea urchin mitochondrial D-loop binding protein (mtDBP) is a transcription termination factor that is able to arrest bidirectionally mitochondrial RNA chain elongation. The observation that the mtDBP binding site in the main non-coding region is located in correspondence of the 3' end of the triplex structure, where the synthesis of heavy strand mitochondrial (mt) DNA is either prematurely terminated or allowed to continue, raised the question whether mtDBP could also regulate mtDNA replication. By using a helicase assay in the presence of the replicative helicase of SV40, we show that mtDBP is able to inhibit the enzyme thus acting as a contrahelicase. The impairing activity of mtDBP is bidirectional as it is independent of the orientation of the protein binding site. The inhibition is increased by the presence of the guanosine-rich sequence that flanks mtDBP binding site. Finally, a mechanism of abrogation of mtDBP contrahelicase activity is suggested that is based on the dissociation of mtDBP from DNA caused by the passage of the RNA polymerase through the protein-DNA complex. All these findings favour the view that mtDBP, besides serving as transcription termination factor, could also act as a negative regulator of mtDNA synthesis at the level of D-loop expansion.

An ab initio quantum mechanics calculation that correlates with ligand orientation and DNA cleavage site selectivity in camptothecin-DNA-topoisomerase I ternary cleavage complexes.

Camptothecin (CPT), a cytotoxic natural alkaloid isolated from Camptotheca acuminata, and its derivatives represent an important class of cancer chemotherapeutic drugs that act by inhibiting topoisomerase I (top1). The mechanism of top1 inhibition by CPT has been determined by X-ray crystallography. Biochemical studies carried out both in vitro and in vivo indicated that CPT has strict DNA sequence preference for -1 T and strong preference for +1 G at the cleavage site. To understand the molecular determinants for the CPT binding orientation and DNA sequence selectivity, we present a quantum mechanics calculation where only pi-pi stacking interactions were included to shed some light on the mechanism of this sequence selectivity. This ab initio calculation can not only reproduce the experimental binding orientation of CPT at the cleavage site but also shows very good correlation between the binding energy for different sequences and the observed frequency of CPT-stabilized sites in the SV40 viral genome. Therefore, it can be concluded that hydrogen bonding of the ligand to the surrounding amino acid residues of the protein is of minor significance. The present method should be applicable to other polycyclic top1 inhibitors.

Assembly of the replication initiation complex on SV40 origin DNA.

The assembly of the complex that forms over the simian virus 40 origin to initiate DNA replication is not well understood. This complex is composed of the virus-coded T antigen and three cellular proteins, replication protein A (RPA), DNA polymerase alpha/primase (pol/prim) and topoisomerase I (topo I) in association with the origin. The order in which these various proteins bind to the DNA was investigated by performing binding assays using biotinylated origin DNA. We demonstrate that in the presence of all four proteins, pol/prim was essential to stabilize the initiation complex from the disruptive effects of topo I. At the optimal concentration of pol/prim, topo I and RPA bound efficiently to the complex, although pol/prim itself was not detected in significant amounts. At higher concentrations less topo I was recruited, suggesting that DNA polymerase is an important modulator of the binding of topo I. Topo I, in turn, appeared to be involved in recruiting RPA. RPA, in contrast, seemed to have little or no effect on the recruitment of the other proteins to the origin. These and other data suggested that pol/prim is the first cellular protein to interact with the double-hexameric T antigen bound to the origin. This is likely followed by topo I and then RPA, or perhaps by a complex of topo I and RPA. Stoichiometric analysis of the topo I and T antigen present in the complex suggested that two molecules of topo I are recruited per double hexamer. Finally, we demonstrate that DNA has a role in recruiting topo I to the origin.

Hepatitis C virus NS3 and simian virus 40 T antigen helicases displace streptavidin from 5'-biotinylated oligonucleotides but not from 3'-biotinylated oligonucleotides: evidence for directional bias in translocation on single-stranded DNA.

Helicases are enzymes that use energy from nucleoside triphosphate hydrolysis to unwind double-stranded (ds) DNA, a process vital to virtually every phase of DNA metabolism. Helicases have been classified as either 5'-to-3' or 3'-to-5' on the basis of their ability to unwind duplex DNA adjacent to either a 5' or 3' single-stranded (ss) DNA overhang. However, there has been debate as to whether this substrate preference is indicative of unidirectional translocation on ssDNA. We developed an assay that monitors the ability of a helicase to displace streptavidin from biotinylated oligonucleotides [Morris, P. D., and Raney, K. D. (1999) Biochemistry 38, 5164-5171]. Two helicases identified as having 5'-to-3' polarity displaced streptavidin from the 3'-end of biotinylated oligonucleotides but not from the 5'-end. We performed similar experiments using the 3'-to-5' helicases from the hepatitis C virus (NS3) and SV40 virus (SV40 T antigen). NS3 and SV40 T antigen were able to displace streptavidin from a 5'-biotinylated oligonucleotide but not from a 3'-biotinylated oligonucleotide. NS3 and SV40 T antigen enhanced the spontaneous rate of dissociation of streptavidin from biotin 340-fold and 1700-fold, respectively. The ssDNA binding protein, gp32, did not enhance dissociation of streptavidin from either end of an oligonucleotide. For NS3, the rate of displacement was faster from a 5'-biotinylated 60mer than from a 5'-biotinylated 30mer. The strong directional bias in streptavidin displacement activity exhibited by each helicase is consistent with a directional bias in translocation on ssDNA. The dependence of the reaction with NS3 on the oligonucleotide length suggests that multiple NS3 monomers are necessary for optimal activity.

FlashPlate scintillation proximity assays for characterization and screening of DNA polymerase, primase, and helicase activities.

DNA replication proteins represent a class of extremely well-established anti-infective drug targets for which improvements in assay technology are required in order to support enzyme characterization, HTS, and structure-activity relationship studies. Replication proteins are conventionally assayed using precipitation/filtration or gel-based techniques, and are not yet all suitable for conversion into homogeneous fluorescence-based formats. We have therefore developed radiometric assays for these enzymes based upon FlashPlate technology that can be applied to a wide range of targets using a common set of reagents. This approach has allowed the rapid characterization of DNA polymerase, DNA primase, and DNA helicase activities. The resultant 96-/384-well microplate assays are suitable for primary HTS, hit selectivity determination, and/or elucidating the mechanism of action of inhibitors. In all cases, biotinylated DNA oligonucleotide substrates were tethered to streptavidin-coated scintillant-embedded FlashPlate wells. Various adaptations were employed for each enzyme activity. For DNA polymerase, a short complementary oligonucleotide primer was annealed to the longer tethered oligonucleotide, and polymerization was measured by incorporation of [(3)H]-dNTPs onto the growing primer 3' end. For DNA primase, direct synthesis of short oligoribonucleotides complementary to the tethered DNA strand was measured by incorporation of [(3)H]-rNTPs or by subsequent polymerase extension with [(3)H]-dNTPs from unlabeled primers. For DNA helicase, unwinding of a [(33)P]-labeled oligonucleotide complementary to the tethered oligonucleotide was measured. This robust and flexible system has a number of substantial advantages over conventional assay techniques for this difficult class of enzymes.