title: Kinetic analysis of reverse transcriptase activity of bacterial family A DNA polymerases.
abstract: Some bacterial thermostable, wild-type or genetically engineered family A DNA polymerases have reverse transcriptase activity. However, difference in reverse transcriptase activities of family A DNA polymerases and retroviral reverse transcriptases (RTs) is unclear. In this study, comparative kinetic analysis was performed for the reverse transcriptase activities of the wild-type enzyme of family A DNA polymerase (M1pol(WT)) from Thermus thermophilus M1 and the variant enzyme of family A DNA polymerase (K4pol(L329A)), in which the mutation of Leu329→Ala is undertaken, from Thermotoga petrophila K4. In the incorporation of dTTP into poly(rA)-p(dT)(45), the reaction rates of K4pol(L329A) and M1pol(WT) exhibited a saturated profile of the Michaelis-Menten kinetics for dTTP concentrations but a substrate inhibition profile for poly(rA)-p(dT)(45) concentrations. In contrast, the reaction rates of Moloney murine leukemia virus (MMLV) RT exhibited saturated profiles for both dTTP and poly(rA)-p(dT)(45) concentrations. This suggests that high concentrations of DNA-primed RNA template decrease the efficiency of cDNA synthesis with bacterial family A DNA polymerases.
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan. yasukawa@kais.kyoto-u.ac.jp
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Yasukawa
authors.forename: Atsushi
authors.initials: A
authors.lastname: Konishi
authors.forename: Mayu
authors.initials: M
authors.lastname: Shinomura
authors.forename: Eriko
authors.initials: E
authors.lastname: Nagaoka
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Fujiwara
mesh_terms: D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008979:Moloney murine leukemia virus / Q000201:enzymology; D009154:Mutation; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016963:Thermus thermophilus / Q000201:enzymology*; D013942:Thymine Nucleotides / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D013942:Thymine Nucleotides; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; C024157:thymidine 5'-triphosphate