Cooperative assembly of the bovine papilloma virus E1 and E2 proteins on the replication origin requires an intact E2 binding site.
Using quantitative gel retardation assays the properties of the bovine papilloma virus (BPV) origin recognition protein E1 and the effect of the viral E2 protein on the binding of E1 to BPV origin DNA were examined. As reported previously (Seo, Y.S., Mueller, F., Lusky, M., Gibbs, E., Kim, H.-Y., Phillips, B. and J. Hurwitz (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2865-2869), the E1 protein binds specifically to DNA sequences within the BPV origin (ori+) of replication. We also show that the presence of MgCl2 and ATP could stabilize the E1 ori+ DNA complex. At low levels of E1, ori+ DNA binding was greatly stimulated by the viral E2 protein when the intact E2 binding site 12 was present on the DNA. In addition DNA-protein complexes formed in the presence of both E1 and E2 were more stable than those formed with E1 alone. In the absence of an E2 binding site the E2 protein inhibited the binding of E1 to the BPV origin. Spacing of 0 or 9 base pairs between the E1 binding site and the E2 binding site 12 abolished the stimulation of E1-DNA binding by E2, whereas spacing of 6 base pairs between the two binding sites allowed for efficient stimulation. The data presented account for a direct role of E2 in BPV DNA replication. We propose that the cooperative binding of both the E1 and E2 proteins to BPV ori+ DNA is mediated by protein-protein interactions and by protein-DNA interactions, which include the formation of specific contacts of E2 with DNA.
Lusky
M
M
Cornell University Graduate School of Medical Sciences, Hearst Microbiology Research Center, Department of Microbiology New York, New York 10021.
Hurwitz
J
J
Seo
Y S
YS
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D052688:Bovine papillomavirus 1 / Q000378:metabolism; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D014764:Viral Proteins / Q000378:metabolism*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; C069814:E1 protein, Bovine papillomavirus; C051550:E2 protein, Bovine papillomavirus; D014764:Viral Proteins; D000255:Adenosine Triphosphate