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Proofreading01:43

Proofreading

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Overview
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Proofreading01:31

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Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore,  it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
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Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
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Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
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A mutation is a change in the sequence of bases of DNA or RNA in a genome. Some mutations occur during replication of the genome due to errors made by the polymerase enzymes that replicate DNA or RNA. Unlike DNA polymerase, RNA polymerase is prone to errors because it is not capable of “proofreading” its work. Viruses with RNA-based genomes, like HIV, therefore accrue mutations faster than viruses with DNA-based genomes. Because mutation and recombination provide the raw material...
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Bacterial Transcription01:53

Bacterial Transcription

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RNA polymerase (RNAP) carries out DNA-dependent RNA synthesis in both bacteria and eukaryotes. Bacteria do not have a membrane-bound nucleus. So, transcription and translation occur simultaneously, on the same DNA template.
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Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis
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Synthetic evolutionary origin of a proofreading reverse transcriptase.

Jared W Ellefson1, Jimmy Gollihar2, Raghav Shroff2

  • 1Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas, 2500 Speedway, Austin, TX 78712, USA. jaredwellefson@gmail.com ellingtonlab@gmail.com.

Science (New York, N.Y.)
|June 25, 2016
PubMed
Summary
This summary is machine-generated.

Scientists evolved a new enzyme, reverse transcription xenopolymerase (RTX), that accurately copies RNA into DNA. This proofreading enzyme overcomes limitations of traditional reverse transcriptase, enabling advanced molecular biology applications.

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Area of Science:

  • Molecular Biology
  • Enzymology
  • Evolutionary Biology

Background:

  • Most reverse transcriptase (RT) enzymes lack proofreading domains, leading to high error rates.
  • This lack of fidelity is a significant limitation in molecular biology applications.

Purpose of the Study:

  • To investigate if proofreading is functionally compatible with reverse transcription.
  • To evolve a high-fidelity DNA polymerase capable of efficient RNA templating.

Main Methods:

  • Directed evolution of a thermostable DNA polymerase.
  • Testing the engineered enzyme's activity on both DNA and RNA templates.
  • Assessing proofreading capability using DNA and RNA substrates.

Main Results:

  • Successfully evolved a novel enzyme, reverse transcription xenopolymerase (RTX).
  • RTX exhibits active proofreading on both DNA and RNA templates, significantly enhancing fidelity.
  • RTX enables novel applications like direct RNA sequencing and single-enzyme RT-PCR.

Conclusions:

  • Proofreading is compatible with reverse transcription.
  • The engineered RTX enzyme overcomes historical limitations of RT fidelity.
  • RTX opens new avenues for sensitive and accurate molecular diagnostics and research.