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Related Concept Videos

Proofreading01:31

Proofreading

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.
Errors During Replication are Corrected by the DNA Polymerase Enzyme
Proofreading01:43

Proofreading

Synthesis of new DNA molecules starts when DNA polymerase links nucleotides together in a sequence that is complementary to the template DNA strand. DNA polymerase has a higher affinity for the correct base to ensure fidelity in DNA replication. The DNA polymerase furthermore proofreads during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.Errors during Replication Are Corrected by the DNA Polymerase EnzymeGenomic DNA is synthesized in...
Translesion DNA Polymerases02:10

Translesion DNA Polymerases

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.
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
Mismatch Repair01:36

Mismatch Repair

Overview
Mismatch Repair01:20

Mismatch Repair

Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
The human genome has more than 3 billion base pairs of DNA per cell. Prior to cell division, that vast amount of genetic...
Mismatch Repair01:36

Mismatch Repair

Overview

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Related Experiment Video

Updated: Jun 12, 2026

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis
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Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

Published on: June 19, 2018

DNA polymerase beta ribonucleotide discrimination: insertion, misinsertion, extension, and coding.

Nisha A Cavanaugh1, William A Beard, Samuel H Wilson

  • 1Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709-2233, USA.

The Journal of Biological Chemistry
|June 4, 2010
PubMed
Summary
This summary is machine-generated.

DNA polymerases discriminate against ribonucleotides, crucial for DNA synthesis. Sugar conformation, specifically the 3'-endo pucker, dictates nucleotide insertion efficiency.

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DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis
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Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis
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Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis
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DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

Published on: October 6, 2017

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Enzymology

Background:

  • DNA polymerases select correct nucleotides based on base pairing and sugar structure.
  • Cells contain higher concentrations of ribonucleotides than deoxynucleotides, posing a challenge for DNA replication fidelity.
  • Therapeutic nucleotide analogs often feature sugar modifications, impacting their efficacy in DNA synthesis.

Purpose of the Study:

  • To investigate the substrate specificity of DNA polymerase beta regarding modified nucleotide sugars.
  • To understand the role of sugar conformation in nucleotide insertion and DNA synthesis.

Main Methods:

  • Assessing the insertion efficiency of various nucleotides, including ribonucleotides and arabinofuranosylcytosine triphosphate, by DNA polymerase beta.
  • Evaluating the impact of templating ribonucleotides on polymerase activity.
  • Analyzing the influence of primer terminus sugar pucker on nucleotide incorporation.

Main Results:

  • DNA polymerase beta efficiently inserts dideoxynucleoside triphosphates but poorly incorporates ribonucleotides (nearly 4-fold less efficient than deoxynucleotides).
  • Once inserted, ribonucleotides are readily extended, and templating ribonucleotides minimally affect insertion or fidelity.
  • Arabinofuranosylcytosine triphosphate is efficiently inserted but poorly extended, highlighting sugar-specific polymerase activity.

Conclusions:

  • The sugar pucker conformation at the primer terminus is critical for DNA synthesis.
  • A 3 -endo sugar pucker conformation favors nucleotide insertion.
  • A 2 -endo sugar pucker conformation inhibits nucleotide insertion, explaining polymerase discrimination against ribonucleotides.