The papillomavirus E1 protein forms a DNA-dependent hexameric complex with ATPase and DNA helicase activities - PubMed (original) (raw)

The papillomavirus E1 protein forms a DNA-dependent hexameric complex with ATPase and DNA helicase activities

J Sedman et al. J Virol. 1998 Aug.

Abstract

The E1 protein from bovine papillomavirus has site-specific DNA binding activity, DNA helicase activity, and DNA-dependent ATPase activity consistent with the properties of an initiator protein. Here we have identified and characterized a novel oligomeric form of E1 that is associated with the ATPase and DNA helicase activities and whose formation is strongly stimulated by single-stranded DNA. This oligomeric form corresponds to a hexamer of E1.

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Figures

FIG. 1

FIG. 1

Incubation of E1 in the presence of single-stranded DNA and ATP results in the quantitative conversion of E1 into an oligomeric complex. (A) E1 was incubated under conditions used for DNA-dependent ATPase assays and was subsequently loaded onto a 15 to 30% glycerol gradient and sedimented as described in Materials and Methods. The gradient was fractionated into 40 fractions, and every other fraction was analyzed for ATPase activity. The glycerol gradient fractions were analyzed for the presence of E1 by Western blotting with a monoclonal antibody directed against E1. ALD, aldolase; CAT, catalase; THR, thyroglobulin. (B) The ATPase activity of the oligomeric E1 complex is not stimulated by DNA. Material from the oligomeric peak in the glycerol gradient in panel A was analyzed for ATPase activity in the absence or presence of poly(dT) over a 60-min time course. In parallel, 0.2 μg of purified, unfractionated E1 protein was analyzed under the same conditions.

FIG. 2

FIG. 2

The oligonucleotide is stably associated with the oligomeric E1 complex. (A) E1 was incubated in the presence of a 27-mer oligonucleotide and sedimented on a gradient as described in the legend to Fig. 1, except that a small fraction of 32P-labeled 27-mer oligonucleotide was added. The gradient fractions were analyzed for the presence of 32P counts. ALD, aldolase; CAT, catalase; THR, thyroglobulin. (B) Western blot analysis of the oligomeric E1 peak. The E1 protein in the oligomer peak was quantitated by comparison to a titration of known quantities (50, 25, 12, 8, 6, and 4 ng) of purified E1 protein. (C) E1 was incubated in the presence of oligonucleotide as described in the legend to Fig. 1, except that 10-fold lower quantities of E1 protein (1.2 μg) and oligonucleotide (0.2 μg) were used, followed by glycerol gradient sedimentation. The fractions were analyzed by Western blotting.

FIG. 3

FIG. 3

Fractions containing the oligomeric E1 complex have DNA helicase activity. (A) Glycerol gradient fractions containing the peak of tracer oligonucleotide were analyzed for DNA helicase activity by an oligonucleotide displacement assay (lanes 1 to 5). Purified E1 (300, 30, or 3 ng) was used in the same assay as a positive control with the same substrate in the presence (lanes 8 to 10) and absence (lanes 11 to 13) of ATP. (B) Western blot of the same gradient fractions that were used in the helicase assay probed with a monoclonal antibody directed against E1.

FIG. 4

FIG. 4

The relative molecular mass of the oligomeric E1 complex is consistent with that of a hexamer of E1. The relative molecular mass of the oligomeric E1 complex was estimated by using a combination of glycerol gradient centrifugation (A) and gel filtration analysis (B) to determine the S value and Stokes’ radius for the oligomeric complex relative to those of marker proteins. The Stokes’ radius for the oligomeric E1 complex was determined to be 82 ± 5 Å, and the sedimentation rate was 12.7S ± 0.5S, resulting in an estimated molecular mass of 420 ± 40 kDa. The marker proteins were BSA (35 Å; 4.2S), aldolase (ALD) (46 Å; 8.3S), catalase (CAT) (52 Å; 11.3S), and thyroglobulin (THR) (85 Å; 18.5S).

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