Reverse transcriptase of mouse mammary tumour virus: expression in bacteria, purification and biochemical characterization.
We have constructed a plasmid that induces in bacteria the synthesis of an enzymically active reverse transcriptase (RT) of mouse mammary tumour virus (MMTV), a retrovirus with a typical B-type morphology. The highest catalytic activity was detected only when 27 residues from the C-terminus of the protease were included in the N-terminus of the recombinant RT, after an extra deoxyadenosine was added between the pro and pol genes to overcome the -1 frameshift event (which occurs naturally in virus-infected cells). The recombinant protein with a six-histidine tag was purified to homogeneity by a two-column purification procedure, Ni2+ nitriloacetic acid/agarose followed by carboxymethyl-Sepharose chromatography. Unlike most RTs, the purified MMTV RT is enzymically active as a monomer even after binding a DNA substrate. Like all RTs studied, the recombinant MMTV RT possesses RNA-dependent and DNA-dependent DNA polymerase activities as well as RNase H activity, all of which show a preference for Mg2+ over Mn2+ ions. Other features of these enzymic activities, such as extension of DNA primers, processivity of DNA synthesis, pH dependence, steady-state kinetic constants, effects of Na+ or K+ ions and sensitivity to a thiol-specific reagent and to a zinc chelator, have been evaluated. The catalytic properties of MMTV RT were compared with those of the well-studied RT of HIV-1, the causative agent of AIDS. Interestingly, MMTV RT exhibits a high sensitivity to nucleoside triphosphate analogues (which are known to be potent inhibitors of HIV RTs and are being used as the major anti-AIDS drugs), as high as that of HIV-1 and HIV-2 RTs. Furthermore the recombinant MMTV RT shows a processivity of DNA synthesis higher than that of HIV-1 RT.
Taube
R
R
Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Israel.
Loya
S
S
Avidan
O
O
Perach
M
M
Hizi
A
A
D000818:Animals; D002384:Catalysis; D002413:Cations, Divalent; D002458:Cell Fractionation; D003545:Cysteine / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008324:Mammary Tumor Virus, Mouse / Q000201:enzymology*; D051379:Mice; D019384:Nucleic Acid Synthesis Inhibitors; D010443:Peptide Chain Termination, Translational / Q000187:drug effects; D011189:Potassium Chloride / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D012194:RNA-Directed DNA Polymerase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D016914:Ribonuclease H / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology
D002413:Cations, Divalent; D019384:Nucleic Acid Synthesis Inhibitors; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D012965:Sodium Chloride; D011189:Potassium Chloride; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H; D003545:Cysteine