Characterization of recombinant HPV6 and 11 E1 helicases: effect of ATP on the interaction of E1 with E2 and mapping of a minimal helicase domain.
To better characterize the enzymatic activities required for human papillomavirus (HPV) DNA replication, the E1 helicases of HPV types 6 and 11 were produced using a baculovirus expression system. The purified wild type proteins and a version of HPV11 E1 lacking the N-terminal 71 amino acids, which was better expressed, were found to be hexameric over a wide range of concentrations and to have helicase and ATPase activities with relatively low values for K(m)(ATP) of 12 microm for HPV6 E1 and 6 microm for HPV11 E1. Interestingly, the value of K(m)(ATP) was increased 7-fold in the presence of the E2 transactivation domain. In turn, ATP was found to perturb the co-operative binding of E1 and E2 to DNA. Mutant and truncated versions of in vitro translated E1 were used to identify a minimal ATPase domain composed of the C-terminal 297 amino acids. This fragment was expressed, purified, and found to be fully active in ATP hydrolysis, single-stranded DNA binding, and unwinding assays, despite lacking the minimal origin-binding domain.
White
P W
PW
Department of Biological Sciences, Boehringer Ingelheim (Canada) Ltd., Laval, Quebec H7S 2G5, Canada. pwhite@lav.boehringer-ingelheim.com
Pelletier
A
A
Brault
K
K
Titolo
S
S
Welchner
E
E
Thauvette
L
L
Fazekas
M
M
Cordingley
M G
MG
Archambault
J
J
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001704:Biopolymers; D002384:Catalysis; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D008969:Molecular Sequence Data; D027383:Papillomaviridae / Q000201:enzymology* / Q000302:isolation & purification; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D001704:Biopolymers; D017931:DNA Primers; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004265:DNA Helicases