Efficient N-tailing of blunt DNA ends by Moloney murine leukemia virus reverse transcriptase.
Moloney murine leukemia virus reverse transcriptase (MMLV-RT) is a widely used enzyme for cDNA synthesis. Here we show that MMLV-RT has a strong template-independent polymerase activity using blunt DNA ends as substrate that generates 3' overhangs of A, C, G, or T. Nucleotides were appended efficiently in the order A > G > T > C, and tail lengths varied from 4 to 5, 2 to 7, 2 to 4, and 2 to 3 for A, C, G, and T, respectively. The activity was so strong that nearly all our test DNA ends were appended with at least one A, C, G, or T. The N-tailing activity of MMLV-RT was enhanced in the presence of Mn2+, and the G-, C-, and T-tailing activities were further enhanced by dCMP, dGMP, and dAMP, respectively. This is the first report of an enzymatic activity that almost thoroughly appends two or more As, or one or more Cs, Gs, or Ts to the 3' end of double-stranded DNA, which would enable exhaustive analysis of DNA samples. The N-tailing activity of MMLV-RT is potentially useful in many biotechnological applications.
Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai 980-8577, Japan.
Yoshiyuki
Y
Ohtsubo
Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai 980-8577, Japan.
Yuji
Y
Nagata
Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai 980-8577, Japan.
Masataka
M
Tsuda
D000818:Animals; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics / Q000502:physiology*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D048348:Reverse Transcription; D013698:Templates, Genetic
D018076:DNA, Complementary; D004247:DNA; D012194:RNA-Directed DNA Polymerase