Template‐independent DNA synthesis activity associated with the reverse transcriptase of the long terminal repeat retrotransposon Tf1

Abstract

Reverse transcriptases (RTs) possess a non-templated addition (NTA) activity while synthesizing DNA with blunt-ended DNA primer ⁄ templates. Interestingly, the RT of the long terminal repeat retrotransposon Tf1 has an NTA activity that is substantially higher than that of HIV-1 or murine leukemia virus RTs. By performing steady state kinetics, we found that the differences between the NTA activities of Tf1 and HIV-1 RTs can be explained by the substantially lower K M value for the incoming dNTP of Tf1 RT (while the differences between the apparent k cat values of these two RTs are relatively small). Furthermore, the K M values, calculated for both RTs with the same dNTP, are much lower for the template-dependent synthesis (TDS) than those of NTA. However, TDS of HIV-1 RT is higher than that of Tf1 RT. The overall relative order of the apparent k cat ⁄ K M values for dATP is: HIV-1 RT (TDS) > Tf1 RT (TDS) >> Tf1 RT (NTA) > HIV-1 RT (NTA). Under the employed conditions, Tf1 RT can add up to seven nucleotides to the blunt-ended substrate, while the other RTs add mostly a single nucleotide. The NTA activity of Tf1 RT is restricted to DNA primers. Furthermore, the NTA activity of Tf1 and HIV-1 RTs is suppressed by ATP, as it competes with the incoming dATP (although ATP is not incorporated by the NTA activity of the RTs). The unusually high NTA activity of Tf1 RT can explain why, after completing cDNA synthesis, the in vivo generated Tf1 cDNA has relatively long extra sequences beyond the highly conserved CA at its 3¢-ends.

Comparison of RNA- or LNA-hybrid oligonucleotides in template-switching reactions for high-speed sequencing library preparation

“…We therefore hypothesize that the RNA template-switching oligonucleotides are more efficient with capped templates. A key difference is that capped templates induce the reverse-transcriptase to extend the first-strand cDNA with cytosines because it reverse-transcribes the cap [16-18], while with non-capped templates, extensions are more rare [17], or of a different nature [19,20]. We speculate that only the presence of DNA/RNA base pairs facilitate template-switching at capped ends, and that DNA-DNA, DNA-LNA and DNA-RNA oligonucleotides have the same efficiency on blunt RNA/cDNA hybrids.…”

Substrate variations that affect the nucleic acid clamp activity of reverse transcriptases

“…BIV RT also has significant NTA activity, but lower than that of Tf1 RT (data not shown). HIV‐1 RT also shows NTA activity, but considerably lower than that for Tf1 and BIV RTs, while MLV RT shows hardly any NTA activity [10–12]. Therefore, some second‐cycle products of clamp/synthesis were also observed with BIV RT (Figs 2A and 3), but no significant activity was detected with HIV‐1 and MLV RTs in any of the experiments (Figs 1–3).…”

“…1, lanes 3 and 4). This results from the high non‐templated addition (NTA) (or terminal deoxynucleotide transferase) activity that is typical of Tf1 RT [10,11]. Thus, the 59‐nucleotide blunt‐ended products of the first combined clamp/polymerase activity cycle are extended by a few nucleotides due to this DNA synthesis activity of Tf1 RT.…”

The Long Terminal Repeat Retrotransposons Tf1 and Tf2 of Schizosaccharomyces pombe

“…Interestingly, the PPT removal activity of RT is greatly stimulated by IN (40). Other biochemical assays demonstrate that Tf1 RT possesses several unusual activities such as template-independent DNA synthesis and the ability to clamp very short primer terminal sequences onto the 3′ termini of template strands (41)(42)(43)(44).…”