title: Selective inactivation of M-MuLV RT RNase H activity by site-directed PEGylation: an improved ability to synthesize long cDNA molecules.
abstract: Moloney murine leukemia virus reverse transcriptase (M-MuLV RT) is a domain structured enzyme that has the N-terminally located DNA polymerization activity and C-terminally located RNase H activity, which interferes with the efficient synthesis of long cDNA molecules. Here we present the PEGylation as a tool for engineering the M-MuLV RT derivative deficient in RNase H activity. We demonstrate that site-directed chemical modification (SDCM) of the RNase H domain by selectively PEGylating C635, one of the eight cysteine residues present in the reverse transcriptase (RT), specifically inactivated its ribonucleolytic activity. As a consequence, the efficiency of long cDNA molecules synthesis by modified enzyme was greatly increased.
authors.affiliation: Thermo Fisher Scientific (formerly Fermentas), Graiciuno 8, Vilnius LT-02241, Lithuania.
authors.forename: Tomas
authors.initials: T
authors.lastname: Radzvilavicius
authors.forename: Arunas
authors.initials: A
authors.lastname: Lagunavicius
mesh_terms: D000818:Animals; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000737:chemistry; D051379:Mice; D008979:Moloney murine leukemia virus / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry*; D048348:Reverse Transcription; D016914:Ribonuclease H / Q000037:antagonists & inhibitors / Q000737:chemistry*
chemical_list: D018076:DNA, Complementary; D011092:Polyethylene Glycols; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H