title: Mutations to create thermostable reverse transcriptase with bacterial family A DNA polymerase from Thermotoga petrophila K4.
abstract: Family A DNA polymerase (K4PolI) from Thermotoga petrophila K4 was obtained as a recombinant form, and the enzyme characteristics were analyzed. K4PolI showed thermostable DNA-dependent DNA polymerase activity with 3'-5' exonuclease activity but no detectable RNA-dependent DNA polymerase activity. Its tertiary structure was speculated by in silico modeling to understand the binding situation between K4PolI and template DNA. Nine amino acids in the 3'-5' exonuclease domain are predicted to be involved in DNA/RNA distinction by steric interference with the 2' hydroxy group of ribose. To allow K4PolI to accept RNA as the template, mutants were constructed focusing on the amino acids located around the 2' hydroxyl group of the bound ribose. The mutants in which Thr326, Leu329, Gln384, Phe388, Met408, or Tyr438 was replaced with Ala (designated as T326A, L329A, Q384A, F388A, M408A, or Y438A, respectively) showed RNA-dependent DNA polymerase activity. All the mutants showed reduced 3'-5' exonuclease activity, suggesting that gain of reverse transcriptase activity is correlated with loss of 3'-5' exonuclease activity. In particular, the mutants enabled direct DNA amplification in a single tube format from structured RNA that was not efficiently amplified by retroviral reverse transcriptase.
authors.affiliation: Department of Bioscience, Graduate School of Science and Technology, Kwansei-Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan.
authors.forename: Sotaro
authors.initials: S
authors.lastname: Sano
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Yamada
authors.forename: Tomoki
authors.initials: T
authors.lastname: Shinkawa
authors.forename: Satoru
authors.initials: S
authors.lastname: Kato
authors.forename: Takashi
authors.initials: T
authors.lastname: Okada
authors.forename: Hiroki
authors.initials: H
authors.lastname: Higashibata
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Fujiwara
mesh_terms: D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005092:Exonucleases / Q000235:genetics / Q000378:metabolism; D006089:Gram-Negative Anaerobic Bacteria / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D017931:DNA Primers; D012336:RNA, Ribosomal, 16S; D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases