The catalytic properties of human hepatitis B virus polymerase.
The DNA-dependent DNA polymerase (DDDP) and RNA-dependent DNA polymerase (RDDP) activities of hepadnavirus polymerases are both essential for viral replication. Human hepatitis B virus (HBV) polymerase has been successfully expressed in Escherichia coli as a fusion protein in frame with maltose-binding protein. The present study was undertaken to characterize these two activities and introduce an in vitro assay system. In situ activity gel assays show that the polymerase has both types of activities. One hundred thirty-four kilodaltons of active full-length product was proteolytically cleaved into approximately 73 kDa of active fragment by proteinase K preincubation. Mutation of conserved YMDD motif also confirms that the activities were due to the recombinant polymerase and that this motif is essential to polymerase activity. Two activities of the polymerase show their optima under conditions of 1 mM (DDDP) or 0.25 mM (RDDP) of MnCl2, 400 mM KCl, 37 degrees C (DDDP) or 24 degrees C (RDDP), and pH 7.0-7.7. Substitution of Mg2+ for Mn2+ results in reduction of processivity, which may explain why Mn2+ supports nucleotide incorporation to a higher level than Mg2+. The polymerase is resistant to aphidicolin. Actinomycin D acts selectively on DDDP activity, whereas phosphonoformic acid inhibits both activities. The in vitro HBV polymerase assay system demonstrated herein will be useful for screening potential HBV polymerase inhibitor for the development of anti-HBV drugs.
Jeong
J H
JH
Deparmtent of Biology Education and Research Center for Cell Differentiation, College of Education, Seoul National University, Korea.
Kwak
D S
DS
Rho
H M
HM
Jung
G
G
D000595:Amino Acid Sequence; D016590:Aphidicolin / Q000494:pharmacology; D001665:Binding Sites; D002413:Cations, Divalent / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D017124:Conserved Sequence; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D003609:Dactinomycin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017245:Foscarnet / Q000494:pharmacology; D006515:Hepatitis B virus / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D017895:Manganese Compounds / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012194:RNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D002413:Cations, Divalent; D002712:Chlorides; D004791:Enzyme Inhibitors; D017895:Manganese Compounds; D011994:Recombinant Proteins; D015636:Magnesium Chloride; D003609:Dactinomycin; D017245:Foscarnet; D016590:Aphidicolin; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; C025340:manganese chloride