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Soluble Rous sarcoma virus reverse transcriptases alpha, alphabeta, and beta purified from insect cells are processive DNA polymerases that lack an RNase H 3' --> 5' directed processing activity.


J Biol Chem. 1999 Sep 10;274(37):26329-36. Unique Identifier : AIDSLINE

Reverse transcriptase (RT) isolated from Rous sarcoma virus (RSV) consists of heterodimeric RTalphabeta, RTalpha, and RTbeta. The alpha subunit (63 kDa) contains an N-terminal polymerase and a C-terminal RNase H domain. The N terminus of beta (95 kDa) corresponds to alpha with the integrase domain attached to the C terminus (32 kDa). We have constructed baculoviruses expressing the genes for alpha or beta or the entire pol (99 kDa). Infection of insect cells with recombinant virus yielded highly active and soluble RSV RT enzymes that could be purified to >90% homogeneity. HPLC gel filtration showed that alpha is a dimeric enzyme that can be partially monomerized upon the addition of 45% Me(2)SO. DNA synthesis on DNA-DNA and DNA-RNA primer-templates in the presence of competitor substrates revealed that alphabeta and beta as well as alpha are processive polymerases. However, the affinity of beta and alphabeta for primer-template substrates appears to be higher than that of alpha. All RSV enzymes investigated have the potential to displace RNA-RNA duplexes more efficiently than human immunodeficiency virus type 1 RT. Unlike human immunodeficiency virus type 1 RT, RSV RTs can catalyze an initial RNase H endonucleolytic cleavage of the RNA template but not a 3' --> 5' directed processing activity.

JOURNAL ARTICLE Animal Chromatography, Gel Chromatography, High Pressure Liquid DNA-Directed DNA Polymerase/CHEMISTRY/GENETICS/*METABOLISM Electrophoresis, Polyacrylamide Gel Hydrolysis Isoenzymes/CHEMISTRY/ISOLATION & PURIF/*METABOLISM Protein Conformation Protein Processing, Post-Translational Recombinant Proteins/CHEMISTRY/ISOLATION & PURIF/METABOLISM Ribonuclease H, Calf Thymus/*METABOLISM RNA-Directed DNA Polymerase/CHEMISTRY/ISOLATION & PURIF/ *METABOLISM Sarcoma Viruses, Avian/*ENZYMOLOGY Spodoptera Support, Non-U.S. Gov't


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