Lk. Thorner et al., DNA-BINDING DOMAIN OF BOVINE PAPILLOMAVIRUS TYPE-1 E1 HELICASE - STRUCTURAL AND FUNCTIONAL-ASPECTS, Journal of virology, 67(10), 1993, pp. 6000-6014
The E1 protein of bovine papillomavirus type 1 is a multifunctional en
zyme required for papillomaviral DNA replication. It assists in the in
itiation of replication both as a site-specific DNA-binding protein an
d as a DNA helicase. Previous work has indicated that at limiting E1 c
oncentrations, the E2 protein is required for efficient E1 binding to
the replication origin. In this study, we have defined the domain of t
he E1 protein required for site-specific DNA binding. Experiments with
a series of truncated proteins have shown that the first amino-termin
al 299 amino acids contain the DNA-binding domain; however, the coterm
inal M protein, which is homologous to E1 for the first 129 amino acid
s, does not bind origin DNA. A series of small internal deletions and
substitution mutations in the DNA-binding domain of E1 show that speci
fic 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 subdomai
n is also important for DNA binding. A region of E1 that interacts wit
h 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 prote
ins were expressed from mammalian expression vectors to ascertain whet
her site-specific DNA binding by E1 is required for transient DNA repl
ication in the cell. These E1 proteins display a range of mutant pheno
types, consistent with the suggestion that site-specific binding by E1
is important. Interestingly, one E1 mutant which is defective for ori
gin binding but can be rescued for such activity by E2 supports signif
icant replication in the cell.