Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In an effort to improve reverse transcriptase (RT) fidelity, we measured the error rate of Moloney murine leukemia virus (MMLV) RT in the presence of several autonomous and DNA polymerase-associated 3'-5' exonucleases using a lacZ forward mutation assay. A number of 3'-5' exonucleases were found to lower the error rate of MMLV RT, including p53, Escherichia coli DNA polymerase III epsilon subunit, and the proofreading activities associated with T4, varphi29, and E. coli pol I DNA polymerases. The bacterial epsilon subunit increased RNA-dependent DNA synthesis fidelity by approximately threefold and was the only 3'-5' exonuclease tested that did not deleteriously affect RT-PCR yields. Further testing showed that RT-PCR mutant frequencies were reduced significantly by performing cDNA synthesis in the presence of epsilon subunit, followed by PCR with a high-fidelity proofreading DNA polymerase. DNA sequence analysis was used to show that the combination of MMLV RT/epsilon subunit and PfuUltra DNA polymerase produces approximately eightfold fewer errors compared with the commonly used combination of MMLV RT and a Taq-based high-fidelity blend, consistent with predictions based on experimentally determined polymerase error rates.
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PMID:Escherichia coli DNA polymerase III epsilon subunit increases Moloney murine leukemia virus reverse transcriptase fidelity and accuracy of RT-PCR procedures. 1710 51

Family A DNA polymerase (K4PolI) from Thermotoga petrophila K4 was obtained as a recombinant form, and the enzyme characteristics were analyzed. K4PolI showed thermostable DNA-dependent DNA polymerase activity with 3'-5' exonuclease activity but no detectable RNA-dependent DNA polymerase activity. Its tertiary structure was speculated by in silico modeling to understand the binding situation between K4PolI and template DNA. Nine amino acids in the 3'-5' exonuclease domain are predicted to be involved in DNA/RNA distinction by steric interference with the 2' hydroxy group of ribose. To allow K4PolI to accept RNA as the template, mutants were constructed focusing on the amino acids located around the 2' hydroxyl group of the bound ribose. The mutants in which Thr326, Leu329, Gln384, Phe388, Met408, or Tyr438 was replaced with Ala (designated as T326A, L329A, Q384A, F388A, M408A, or Y438A, respectively) showed RNA-dependent DNA polymerase activity. All the mutants showed reduced 3'-5' exonuclease activity, suggesting that gain of reverse transcriptase activity is correlated with loss of 3'-5' exonuclease activity. In particular, the mutants enabled direct DNA amplification in a single tube format from structured RNA that was not efficiently amplified by retroviral reverse transcriptase.
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PMID:Mutations to create thermostable reverse transcriptase with bacterial family A DNA polymerase from Thermotoga petrophila K4. 2214 68

Viral metagenomic libraries are a promising but previously untapped source of new reagent enzymes. Deep sequencing and functional screening of viral metagenomic DNA from a near-boiling thermal pool identified clones expressing thermostable DNA polymerase (Pol) activity. Among these, 3173 Pol demonstrated both high thermostability and innate reverse transcriptase (RT) activity. We describe the biochemistry of 3173 Pol and report its use in single-enzyme reverse transcription PCR (RT-PCR). Wild-type 3173 Pol contains a proofreading 3'-5' exonuclease domain that confers high fidelity in PCR. An easier-to-use exonuclease-deficient derivative was incorporated into a PyroScript RT-PCR master mix and compared to one-enzyme (Tth) and two-enzyme (MMLV RT/Taq) RT-PCR systems for quantitative detection of MS2 RNA, influenza A RNA, and mRNA targets. Specificity and sensitivity of 3173 Pol-based RT-PCR were higher than Tth Pol and comparable to three common two-enzyme systems. The performance and simplified set-up make this enzyme a potential alternative for research and molecular diagnostics.
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PMID:Thermostable DNA polymerase from a viral metagenome is a potent RT-PCR enzyme. 2267 52


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