Synthetic evolutionary origin of a proofreading reverse transcriptase

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 functio...

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Bibliographic Details
Published in:Science (American Association for the Advancement of Science) 2016-06, Vol.352 (6293), p.1590-1593
Main Authors: Ellefson, Jared W., Gollihar, Jimmy, Shroff, Raghav, Shivram, Haridha, Iyer, Vishwanath R., Ellington, Andrew D.
Format: Article
Language:English
Subjects:
Biological evolution
Deoxyribonucleic acid
Directed Molecular Evolution
DNA Mismatch Repair
DNA Mutational Analysis
DNA polymerase
DNA, Complementary - biosynthesis
Enzymes
Evolution, Molecular
Evolutionary
Exonucleases - chemistry
Fidelity
Models, Molecular
Origins
Phylogeny
Proofreading
Protein Structure, Tertiary
Proteins
Pyrococcus furiosus - enzymology
Ribonucleic acid
Ribonucleic acids
RNA
RNA - chemistry
RNA - genetics
RNA-Directed DNA Polymerase - chemistry
RNA-Directed DNA Polymerase - classification
RNA-Directed DNA Polymerase - genetics
Templates, Genetic
Thermococcus - enzymology
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Summary: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.
ISSN:0036-8075
1095-9203
DOI:10.1126/science.aaf5409