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

Iris Oz-Gleenberg, Eytan Herzig, Nickolay Voronin, Amnon Hizi*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

We have recently shown that reverse transcriptases (RTs) perform template switches when there is a very short (two-nucleotide) complementarity between the 3' ends of the primer (donor) strand and the DNA or RNA template acceptor strands [Oz-Gleenberg et al. (2011) Nucleic Acids Res39, 1042-1053]. These dinucleotide pairs are stabilized by RTs that are capable of 'clamping' together the otherwise unstable duplexes. This RT-driven stabilization of the micro-homology sequence promotes efficient DNA synthesis. In the present study, we have examined several factors associated with the sequence and structure of the DNA substrate that are critical for the clamp activity of RTs from human immunodeficiency virus type 1 (HIV-1), murine leukemia virus (MLV), bovine immunodeficiency virus (BIV) and the long terminal repeat retrotransposon Tf1. The parameters studied were the minimal complementarity length between the primer and functional template termini that sustains stable clamps, the effects of gaps between the two template strands on the clamp activity of the tested RTs, the effects of template end phosphorylations on the RT-associated clamp activities, and clamp activity with a long 'hairpin' double-stranded primer comprising both the primer and the complementary non-functional template strands. The results show that the substrate conditions for clamp activity of HIV-1 and MLV RTs are more stringent, while Tf1 and BIV RTs show clamp activity under less rigorous substrate conditions. These differences shed light on the dissimilarities in catalytic activities of RTs, and suggest that clamp activity may be a potential new target for anti-retroviral drugs. Reverse-transcriptases can perform template switches with very short complementarities between the 3'-ends of the primer strand and the DNA or RNA template acceptor strands. We have examined here several factors associated with sequence and structure of the DNA substrate, which are critical for the clamp activity of various reverse-transcriptases. We found interesting dissimilarities in the catalytic activities of the enzymes.

Original languageEnglish
Pages (from-to)1894-1903
Number of pages10
JournalFEBS Journal
Volume279
Issue number10
DOIs
StatePublished - May 2012

Keywords

  • nucleic acids clamp activity
  • reverse transcriptases
  • reverse transcription
  • strand transfer
  • substrate variations

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