Study of SV40 large T antigen nucleotide specificity for DNA unwinding

Abstract

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.

Keywords: DNA replication; Nucleotide binding and hydrolysis; Nucleotide specificity for unwinding; Replicative helicase; SV40 large t antigen; SV40 virus.

Fig. 1

Structure of the SV40 LT nucleotide binding pocket and the various pocket mutants. a Crystal structure of the LT hexameric helicase, represented by residues 251–627 in the form of a hexamer bound to six ATP (PDBid: 1SVM). The nucleotide pocket is located at the interface between adjacent monomers as indicated with an outlined box. b A close-up of the nucleotide pocket bound to ATP and Mg⁺⁺. The P-loop is colored in yellow. c, d, e Locations of the polar/charged residues c and hydrophobic residues e chosen for the mutational analysis of the binding pocket are indicated in magenta and green and their mutants are listed in d

Fig. 2

Helicase assay results of LT nucleotide pocket mutants. a, b The helicase activity assay results for the polar/charged residue mutants a and hydrophobic residue mutants b are shown in gel analysis data and quantitative bar charts. The native gels shown are a single representation from a set of three individual experiments. Each lane (A: ATP, T: TTP, U: UTP, C: CTP, and G: GTP) contains 0.5 mM of each respective NTP, 100 fmol of a 3’-6FAM labeled dsDNA substrate, 90 nM of each respective LT mutant. The ─NTP controls undergo the same reaction without NTP; the ssDNA lane contains the non-annealed labeled ssDNA as the unwound ssDNA control. The lower bands indicate the unwound labeled ssDNA strand. The bar graphs represent the average of three individual experiments in the percentage of strand displacement. Changes were calculated via unpaired t-tests and Bonferroni corrected (n = 16) with a p-value of 0.0031 as threshold for statistical significance, noted with ***

Fig. 3

Helicase assay results of the combined double-mutants of hydrophobic residues around the nucleotide pocket. a The assay results of the unwinding activities of LT mutants’ cW393A/cL397A and cL553A/cL557A. Conditions for the helicase assays are the same as described in Fig. 2. b The position of each set of residues in the nucleotide binding pocket with cW393A/cL397A located on α-helix 7 (purple) and cL553A/cL557A on α-helix 13 (orange) of the LT hexameric helicase structure. P-loop is colored in yellow as a point of reference

Fig. 4

Oligomerization assay of LT WT and nucleotide pocket mutants using gel filtration chromatography on Superdex 200 column. a–f Gel filtration profiles of WT, cS430T, tK419A, cW393A, and cL557A without NTP (a) with ATP (b) with ATP + Mg⁺⁺ (c) with TTP (d) with TTP + Mg⁺⁺ (e) and with UTP ± Mg⁺⁺ (f). The assays were performed by analyzing 250 μg of LT protein with or without the mentioned NTP and Mg⁺⁺ using Superdex 200 10/300 GL column. The two peaks represent the hexamer (~344 kDa) and monomer (~57 kDa) of LT. g The percentages of hexamer formation which were calculated based on the areas under each UV absorption peak in panels A–F. “─” indicates that the oligomerization test in the presence of UTP ± Mg⁺⁺ was not performed. The running buffer contained 25 mM Tris pH 8.0, 250 mM NaCl, and 0.5 mM TCEP. Each protein was incubated with 4 mM NTP and 1 mM MgCl₂ where indicated

Fig. 5

Characterization of ATP binding by LT using isothermal titration calorimetry (ITC). a Raw titration curves of WT ± Mg⁺⁺ and different nucleotide pocket mutants in the presence of Mg⁺⁺. Thirteen titrations were measured (μcal/second) for each curve over seventy minutes. b Graphical representation of the trends in total heat ΔQ changes over each injection. c Quantification of ΔQ over each injection. The negative values indicate an exothermic release of heat. The syringe (ATP) and cell (LT protein) concentrations in each experiment were 400uM and 25uM, respectively and the storage buffer contained 25 mM Tris pH 8.0, 250 mM NaCl, 0.5 mM TCEP, and 1 mM MgCl₂ unless otherwise noted

Fig. 6

Characterization of TTP binding by LT using ITC. a Raw titration curves of WT ± Mg⁺⁺ and different nucleotide pocket mutants in the presence of Mg⁺⁺. Thirteen titrations were measured (μcal/second) for each curve over approximately seventy minutes. b Graphical representation of the trends in total heat ΔQ changes over each injection. c Quantification of ΔQ over each injection. The negative values indicate an exothermic release of heat. The syringe (TTP) and cell (LT protein) concentrations in each experiment were 800uM and 25uM, respectively and the storage buffer contained 25 mM Tris pH 8.0, 250 mM NaCl, 0.5 mM TCEP, and 1 mM MgCl₂ unless otherwise noted

Fig. 7

Characterization of UTP binding by LT using ITC. a Raw titration curves of WT ± Mg⁺⁺ and different nucleotide pocket mutants in the presence of Mg⁺⁺. Thirteen titrations were measured (μcal/second) for each curve over approximately 70 or 120 min. b Graphical representation of the trends in total heat ΔQ changes over each injection. c Quantification of ΔQ over each injection. The negative values indicate an exothermic release of heat. The syringe (UTP) and cell (LT protein) concentrations in each experiment were 800uM and 25uM, respectively and the storage buffer contained 25 mM Tris pH 8.0, 250 mM NaCl, 0.5 mM TCEP, and 1 mM MgCl₂ unless otherwise noted

Fig. 8

Structural implications of LT nucleotide specificity. a Residues cP427, cF547, and cP549 (orange) are presented in sticks to demonstrate the rigidity in the spatial arrangement surrounding cS430 (magenta). b The charge-charge interaction between tK419 (magenta) and the -OH group on the sugar molecule of TTP. c The unique hydrophobic lining of nine hydrophobic residues (green), including cW393 and cL557 around base of the bound nucleotide in the nucleotide pocket of LT. d The function of the hydrophobic lining through an induced fit mechanism as illustrated with TTP, in which cW393, cL557 and other hydrophobic residues alter their conformations compared to those in panel-C. TTP and UTP were docked and fitted into the LT nucleotide binding pocket utilizing AutoDock Vina in PyMol. The lowest energy state docking with the correct alignment of the α-β-γ phosphates of the bound NTP was chosen for their respective panels