Expression, purification, and characterization of an active RNase H domain of the hepatitis B viral polymerase.
The replication of the hepatitis B viral DNA genome proceeds through a pregenomic RNA intermediate. This pregenomic RNA subsequently serves as the template for the formation of the viral DNA by the reverse transcriptase activity of the viral P gene product. The P gene product is believed to be a multifunctional enzyme with DNA-dependent DNA polymerase, RNA-dependent DNA polymerase, and RNase H activities. Detailed biochemical studies of this protein have not been performed because of the inability to obtain sufficient amounts of the enzyme from the virus and by the inability to produce the enzyme in heterologous expression systems. The RNase H activity is essential for viral replication and is believed to be responsible for the degradation of the RNA pregenomic intermediate as well as for generating the short RNA primer that is required for DNA second strand synthesis. We have assembled an expression vector which directs the synthesis of a protein that corresponds to the putative RNase H domain of the P gene product and having a carboxyl-terminal polyhistidine tag to facilitate purification. The protein has been expressed in Escherichia coli and purified to yield 1-2 mg of protein/liter of culture. This protein has RNase H activity as defined by its ability to degrade the RNA component of RNA-DNA hybrids but not the DNA component. The RNase H has a basic optimum pH, is active only in the presence of reducing agents, and is dependent on the presence of divalent cations, with magnesium being preferred over manganese.
Wei
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Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond, Virginia 23298-0614, USA.
Peterson
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004229:Dithiothreitol / Q000737:chemistry; D015684:Gene Products, pol / Q000737:chemistry* / Q000378:metabolism; D005814:Genes, Viral; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D011189:Potassium Chloride; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins; D016914:Ribonuclease H / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000737:chemistry; D015678:Viral Structural Proteins / Q000235:genetics
D018076:DNA, Complementary; D015684:Gene Products, pol; C066027:P protein, Hepatitis B virus; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D015678:Viral Structural Proteins; D011189:Potassium Chloride; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H; D008274:Magnesium; D004229:Dithiothreitol