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Synthetic evolutionary origin of a proofreading reverse transcriptase

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Science  24 Jun 2016:
Vol. 352, Issue 6293, pp. 1590-1593
DOI: 10.1126/science.aaf5409

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Making error-free DNA from RNA

DNA polymerase enzymes copy DNA into new strands of identical DNA. Reverse transcriptase (RT) enzymes copy RNA into DNA. Unlike many DNA polymerases, RT enzymes do not have a proofreading function that checks for errors in the newly synthesized DNA. Ellefson et al. use in vitro directed evolution and protein engineering to build an error-correcting RT from a prokaryotic DNA polymerase. The RT “xenopolymerase” shows increased fidelity as compared to natural RTs and should streamline and increase the precision of transcriptomics methods.

Science, this issue p. 1590

Abstract

Most reverse transcriptase (RT) enzymes belong to a single protein family of ancient evolutionary origin. These polymerases are inherently error prone, owing to their lack of a proofreading (3′- 5′ exonuclease) domain. To determine if the lack of proofreading is a historical coincidence or a functional limitation of reverse transcription, we attempted to evolve a high-fidelity, thermostable DNA polymerase to use RNA templates efficiently. The evolutionarily distinct reverse transcription xenopolymerase (RTX) actively proofreads on DNA and RNA templates, which greatly improves RT fidelity. In addition, RTX enables applications such as single-enzyme reverse transcription–polymerase chain reaction and direct RNA sequencing without complementary DNA isolation. The creation of RTX confirms that proofreading is compatible with reverse transcription.

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