Papillomavirus E1 protein binds to and stimulates human topoisomerase I

Abstract

The papillomavirus (PV) E1 helicase plays a direct role in recruiting cellular DNA replication factors, such as replication protein A or polymerase alpha-primase, to replicate PV genomes. Here, E1 is shown to bind to human topoisomerase I and stimulate its relaxation activity up to sevenfold. The interaction between E1 and topoisomerase I was mapped to the E1 DNA binding domain and C terminus. These findings imply a mechanism for the recruitment of topoisomerase I to PV DNA replication forks and for stimulating topoisomerase I to allow for efficient relaxation of the torsional stress induced by replication fork progression.

FIG. 1.

E1 binds topoisomerase I. (a) ELISAs were performed using 3 pmol of topoI as the immobilized protein. Increasing concentrations of the indicated proteins (BPV-1 GST-E1, HPV-11 GST-E1, GST-YY1) were added to the wells. Binding was detected using a polyclonal α-GST antibody followed by a secondary horseradish peroxidase antibody. After developing, the absorbance was measured at 450 nm. GST also did not bind topoI. The results are representative of three separate experiments; the variation (range) is demonstrated by error bars. (b) ELISAs were performed using 3 pmol of GST-E1, BSA, or GST, as the immobilized protein. Increasing concentrations of topoI were added to the wells. Binding was detected using a monoclonal α-topoI antibody followed by a secondary horseradish peroxidase antibody. After developing, the absorbance was measured at 450 nm. The results are representative of three separate experiments; the variation (range) is demonstrated by error bars. (c) GST-E1 coprecipitation experiments were performed in which 10 or 50 μg of GST-E1 or GST was incubated with glutathione Sepharose beads in the presence of 5 mg of 293 cell extract (S100) or 50 μg of purified topoI (the asterisk indicates which panel is from a separate blot). The beads were washed with high-salt Tris-buffered saline (500 mM NaCl) three times and subjected to sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis. Western analysis was performed using a α-topoI monoclonal antibody (the asterisk also indicates the use of a polyclonal antibody).

FIG. 2.

E1 stimulates topoisomerase I relaxation activity. (a) topoI relaxation assays were performed in the presence of increasing levels of GST-E1. Plasmid template pUC119 (30 ng/μl; lane 2) was incubated with 5 pg/μl of topoI (+) for 30 min at 37°C (lane 1). GST-E1 was added to the reaction at 3, 7.5, and 15 ng/μl in lanes 3 through 5. GST-E1 (30 ng/μl) alone had no effect on the plasmid DNA topology (lane 7). GST (30 ng/μl) also had no effect on topoI activity (lane 6). BSA was added to each reaction at 0.1 mg/ml. Arrows show positions of relaxed or nicked (form II/IV) and supercoiled (form I) plasmid DNA. (b) The degree of topoI stimulation was measured by the disappearance of the supercoiled band. topoI stimulation (of DNA relaxation), produced by E1 (and other proteins), was calculated by comparison with a topoI standard curve. Standard curves were created by adding increasing amounts of topoI to relaxation assays in order to produce a quantitative measure of the amount of topoI required to generate a specific amount of relaxed form IV DNA. The addition of E1 to a topoI relaxation assay produced a stimulation of DNA relaxation that was subsequently compared to the standard curve, which then produced a numerical value (n-fold stimulation) correlating to a concentration of topoI needed to achieve the equivalent amount of form I DNA. The concentration of GST-E1 is shown, and all other proteins were assayed at the same molar equivalent. The results are representative of three or more separate experiments; the variation (range) is demonstrated by error bars. (c) The PV origin of replication does not affect the stimulation of topoI by E1. Relaxation assays were performed as described above using a PV origin (pUCAlu, a plasmid containing the BPV-1 origin; nucleotides 7891 through 7952) or a nonorigin (pUC119) template (7, 14). The degree of topoI stimulation on the various templates was measured as described above. The results are representative of three or more separate experiments; the variation (range) is demonstrated by error bars.

FIG. 3.

DBD and C terminus of E1 bind topoI. (a) ELISAs were performed using 3 pmol of immobilized topoI as described above. As second proteins, GST-E1, GST-E1 aa 1 through 403, BPV-1 GST-E1 DBD, GST-E1 aa 1 through 165, and GST alone are depicted. (b) ELISAs were performed using 3 pmol of immobilized topoI. As second proteins, GST-E1, GST-E1 aa 589 through 649, GST-E1 aa 310 through 649, GST-E1 aa 346 through 649, and GST alone are shown. All results represent three separate experiments; the variation (range) is depicted by error bars. (c) E1 binds topoI through two regions (A and B). Graphic depictions of linear representations of E1 showing the binding results in panels a and b. Binding site A is the E1 DBD, and binding site B consists of amino acids 589 through 649 (shaded regions).

FIG. 4.

topoI stimulation by E1 truncations. (a) topoI plasmid relaxation assays were performed in the presence of a second protein: GST, BSA, HPV-11 GST-E1 (aa 589 through 649), BPV-1 GST-E1 DBD (aa 142 through 308), HPV-11 GST-E1 (aa 1 through 165), BPV-1 GST-E1, HPV-11 GST-E1, or HPV-11 GST-E1 (aa 1 through 403) as labeled (lanes 2 and 4 through 10). Plasmid template pUC119 (30 ng/μl) was incubated with 5 pg/μl of topoI (+) for 30 min at 37°C, where indicated. Second proteins were added to the reaction at the same molar equivalent as full-length GST-E1. The reaction shown in lane 1 was performed without a second protein. Lane 3 shows pUC119 with no topoI. Lane 11 shows plasmid with GST-E1 and no topoI. BSA was added to each reaction at 0.1 mg/ml. Arrows show positions of relaxed (form II/IV) and supercoiled (form I) plasmid DNA. (b) Stimulation of topoI relaxation of DNA produced by the proteins and E1 truncations indicated was calculated as described in the legend to Fig. 2. The concentration of GST-E1 is shown, and all other proteins were assayed at the same molar equivalent. Higher levels of the proteins did not show appreciably greater stimulation (data not shown). The results are representative of three or more separate experiments; the variation (range) is shown by error bars.