Understanding the biochemistry of DNA replication and the associated regulation of cell cycle in eukaryotes is difficult at a molecular level due to the complexity of these genomes. As an alternative, small extrachromosomal genomes, such as yeast ARS plasmids and certain DNA viruses, have been explored as model systems for cellular DNA replication and its regulation. Papillomaviruses are an attractive model system because replication of the viral genome is coordinately regulated with the host cell cycle during stable infection, which implies that the viral genome is utilizing normal cellular control mechanisms. The long-range goal of this work will be the identification and characterization of all the cellular and viral proteins required for authentic, regulated papillomavirus replication. As a first step in this process, the immediate goal will be to define the mechanism(s) of action of viral proteins known to be required for replication in vivo. This proposal will focus specifically on the two viral proteins required for replication of bovine papillomavirus (BPV) in vivo, the E1 and E2 proteins. Recent work has shown that the E1 protein binds to BPV DNA in the region that functions as an origin of replication in vivo (Wilson and Ludes-Meyer, manuscript submitted), and that this E1 binding site is adjacent to a binding site for E2 protein (V. Wilson, unpublished observations). Both the E1 and E2 proteins will be expressed in a bacterial system and purified for assays of function in vitro.
Specific aims will be to 1) define the location, organization, and critical nucleotides of the E1 binding site in vitro, 2) determine the effect of E1 binding on BPV DNA structure, 3) establish whether or not origin binding is required for the replication activity of E1 protein, and 4) examine the physical and functional relationship between the E1 and E2 proteins bound at the origin region. These studies should provide the initial information about critical protein-DNA interactions at a functional origin of replication in BPV.
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|Ludes-Meyers, J H; Wilson, V G (1996) Simple procedure for creation of in-frame deletion mutations throughout an open reading frame. Biotechniques 20:433-8|
|Belyavskyi, M; Miller, J; Belyavaskaya, E et al. (1995) BPV E1 protein alters the kinetics of cell cycle entry of serum starved mouse fibroblasts. Cytometry 21:257-64|
|Holt, S E; Wilson, V G (1995) Mutational analysis of the 18-base-pair inverted repeat element at the bovine papillomavirus origin of replication: identification of critical sequences for E1 binding and in vivo replication. J Virol 69:6525-32|
|Belyavskyi, M; Miller, J; Wilson, V (1994) Bovine papillomavirus E1 protein affects the host cell cycle phase fractions. Cytometry 16:129-37|
|Schuller, G; Holt, S E; Hsu, J et al. (1994) The bovine papillomavirus type 1 genome contains multiple loci of static DNA bending, but bends are absent from the functional origin of replication. Virus Res 31:203-17|
|Belyavskyi, M; Miller, J; Wilson, V (1994) The bovine papillomavirus E1 protein alters the host cell cycle and growth properties. Virology 204:132-43|
|Holt, S E; Schuller, G; Wilson, V G (1994) DNA binding specificity of the bovine papillomavirus E1 protein is determined by sequences contained within an 18-base-pair inverted repeat element at the origin of replication. J Virol 68:1094-102|
|Leng, X; Wilson, V G; Xiao, X L (1994) Genetically defined nuclear localization signal sequence of bovine papillomavirus E1 protein is necessary and sufficient for the nuclear localization of E1-beta-galactosidase fusion proteins. J Gen Virol 75 ( Pt 9):2463-7|