DNA-binding domain of bovine papillomavirus type 1 E1 helicase: structural and functional aspects.
The E1 protein of bovine papillomavirus type 1 is a multifunctional enzyme required for papillomaviral DNA replication. It assists in the initiation of replication both as a site-specific DNA-binding protein and as a DNA helicase. Previous work has indicated that at limiting E1 concentrations, the E2 protein is required for efficient E1 binding to the replication origin. In this study, we have defined the domain of the E1 protein required for site-specific DNA binding. Experiments with a series of truncated proteins have shown that the first amino-terminal 299 amino acids contain the DNA-binding domain; however, the coterminal M protein, which is homologous to E1 for the first 129 amino acids, does not bind origin DNA. A series of small internal deletions and substitution mutations in the DNA-binding domain of E1 show that specific basic residues in this region of the protein, which are conserved in all E1 proteins of the papillomavirus family, likely play a direct role in binding DNA and that a flanking conserved hydrophobic subdomain is also important for DNA binding. A region of E1 that interacts with E2 for cooperative DNA binding is also retained in carboxy-terminal truncated proteins, and we show that the ability of full-length E1 to complex with E2 is sensitive to cold. The E1 substitution mutant proteins were expressed from mammalian expression vectors to ascertain whether site-specific DNA binding by E1 is required for transient DNA replication in the cell. These E1 proteins display a range of mutant phenotypes, consistent with the suggestion that site-specific binding by E1 is important. Interestingly, one E1 mutant which is defective for origin binding but can be rescued for such activity by E2 supports significant replication in the cell.
Thorner
L K
LK
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Lim
D A
DA
Botchan
M R
MR
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D052688:Bovine papillomavirus 1 / Q000201:enzymology* / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009036:Moths; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D017354:Point Mutation; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; C069814:E1 protein, Bovine papillomavirus; D011994:Recombinant Proteins; D014764:Viral Proteins; D004265:DNA Helicases