According to the results in Fig. 2b, the supercoiled dsDNA seems to interact with the VP1 protein more than opened dsDNA. Thus, to further address whether the DNA-binding activity of VP1 is affected by DNA conformation, various species of DNA molecules, such as linear dsDNA, circular ssDNA and linear ssDNA were used to confirm VP1 DNA binding activity. Because the DNA binding activity of VP1 has no sequence specificity as illustrated in Fig. 2b, c and d, the commercial circular single-stranded genome of the M13 phage was used as sample DNA instead of the real circular ssDNA genome of CAV. As illustrated in Fig. 4 with respect to all DNA species, DNA retardation occurred when the recombinant VP1 proteins were added to DNA molecules, such as linear pcDNA3.1 (linear dsDNA, Fig. 4a), the linear single strand of the CAV genome (minus and strand, Fig. 4b) and the genome of the M13 phage (circular ssDNA, Fig. 4c). Similarly, with respect to the VP3 protein, all kinds of DNA molecules were also bound by VP3 and reduced the migration of DNA. However, comparing the significance of DNA patterns between various protein-DNA complexes, different DNA molecules bound by VP1 protein demonstrated there were distinct migration patterns of DNA (Fig. 4). Therefore, to address the binding preferences of the VP1 protein to DNA molecules, various amounts of VP1 protein were added to equal amounts of different DNA molecules for analysis of protein-DNA interactions. By quantifying the amount of VP1 protein required for DNA binding with respect to pCAV (circular dsDNA), especially for its supercoiled form dsDNA, the results showed 200 μg of VP1 were required to initiate VP1 binding to DNA molecules (Fig. 5a, supercoiled, form II). At least 300 μg of VP1 were required for this interaction to occur between VP1 and opened dsDNA (Fig. 5a, opened, form I). Higher amounts of VP1 protein were used to bind circular dsDNA and reduced DNA migration patterns more significantly (Fig. 5a). Similarly, other DNA molecules, such as linear dsDNA (linearized pcDNA3.1, Fig. 5b), a linear single strand of the CAV genome (minus strand, Fig. 6a) and circular ssDNA (genome of the M13 phage, Fig. 6b), showed a similar pattern for protein-DNA interactions in the reaction mixture, with approximately 300 μg of VP1 required for linear dsDNA, 200 μg for the minus strand of linear ssDNA and 100 μg for circular ssDNA. In contrast, residual unbound DNA molecules representing the amount of DNA binding on the gel decreased if protein-DNA interaction occurred. Based on these results, the preferences in order of affinity to DNA with the VP1 protein in terms of the estimation of the percentage of unbound DNA molecules, which were sorted from low to high, were circular ssDNA (46.5% with respect to 300 μg of VP1), the linear minus strand of ssDNA (53.3% with respect to 300 μg of VP1), supercoiled circular dsDNA (82.6% with respect to 300 μg of VP1), linear dsDNA (84.7% with respect to 300 μg of VP1) and opened circular dsDNA (100% with respect to 300 μg of VP1). The comparison of binding preferences of VP1 protein to different conformations of DNA molecules are summarized in Table 1. More unbound DNA existed in the agarose gel, implying that this conformation of DNA exhibited a lower preference to interact with the VP1 protein. These results demonstrated that the interaction of VP1 with the DNA molecule exhibited various binding preferences that were dependent on the structural conformation of DNA.Fig. 4VP1 protein binds to various DNA molecules. Purified GST and GST-fused proteins were used for analysing the interaction of recombinant proteins with various DNA samples, such as linear dsDNA (a), minus-strand ssDNA (b), and M13mp18 phage DNA (c). All DNA samples were generated by different preparations as described in the Materials and Methods. After the agarose gel shift assay, the DNA fragment signals were observed by EtBr staining. The 1% SDS (underline lane-labelled 1% SDS) was also used to confirm the retardation caused by tested proteins. Lane M, DNA ladder marker. Bold triangles indicate the protein-DNA complex formed by the tested protein and DNA molecules. The “pcDNA3.1 x EcoR I” indicated generation of the linear form of pcDNA3.1 DNA digested by EcoR IFig. 5Dose-dependent analysis of VP1-dsDNA binding ability. Various concentrations of GST-VP1 were used to perform the dose-dependent analysis with a consistent concentration of plasmid pCAV (a) or linear dsDNA (b). The DNA fragment signals of the protein-DNA complex and differences in DNA migration patterns were more significant as the protein amount increased. Lane M, DNA ladder marker. Bold triangles indicate the protein-DNA complex formed by the tested protein and plasmids. Asterisks indicate the two conformations of plasmid DNAs, including the relaxed form (Form I) and a supercoiled form (Form II). The "pcDNA3.1xEcoRI" indicated generation of the linear form of pcDNA3.1 DNA digested by EcoRI Fig. 6Dose-dependent analysis of VP1-ssDNA binding ability. Increased concentrations of GST-VP1 were incubated with consistent concentrations of minus-strand ssDNA (a) or circular ssDNA (b) to perform dose-dependent analysis. The disappearance of free DNA signals was more obvious when the protein amount increased. Lane M, DNA ladder marker. Bold triangles indicate the protein-DNA complex formed by tested proteins and DNA moleculesTable 1The ratio of unbound free DNA residue was determined by calculating the image intensity of free DNA fragments on an electrophoretic agarose gel after a VP1-DNA binding assay combining increasing amounts of recombinant GST-VP1 protein with certain DNA molecule conformationsConcentrations of GST-VP1 protein (ng)0100300500700Open circular dsDNA (Form I)100%100%100%59.6%11%Supercoiled dsDNA (Form II)100%100%82.6%0%0%Linear dsDNA100%93.8%84.7%36.5%0%Linear ssDNA (−)100%92.9%53.3%0%0%M13 phage DNA100%90%46.5%0%0%The lower ratios indicate a higher preference of the VP1 protein for specific DNA conformations. The equation used in this study is presented under the table