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Related Concept Videos

Proofreading01:31

Proofreading

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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.
Errors During Replication are Corrected by the DNA Polymerase...
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Mismatch Repair01:20

Mismatch Repair

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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...
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The Replisome03:01

The Replisome

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DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with...
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Translesion DNA Polymerases02:10

Translesion DNA Polymerases

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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.
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...
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Lagging Strand Synthesis01:59

Lagging Strand Synthesis

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During replication, the complementary strands in double-stranded DNA are synthesized at different rates. Replication first begins on the leading strand. Replication starts later, occurs more slowly, and proceeds discontinuously on the lagging strand.
There are several major differences between synthesis of the leading strand and synthesis of the lagging strand. 1) Leading strand synthesis happens in the direction of replication fork opening, whereas lagging strand synthesis happens in the...
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Homologous Recombination02:31

Homologous Recombination

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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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Related Experiment Video

Updated: Jun 22, 2025

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

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Kinetic Proofreading Can Enhance Specificity in a Nonenzymatic DNA Strand Displacement Network.

Rakesh Mukherjee1, Aditya Sengar1, Javier Cabello-García2

  • 1Department of Bioengineering, Imperial College London, London SW7 2AZ, U.K.

Journal of the American Chemical Society
|July 1, 2024
PubMed
Summary

This study introduces DNA strand displacement kinetic proofreading to enhance molecular recognition specificity. This method improves how probes detect single nucleotide mutations, boosting accuracy in synthetic systems.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Synthetic Biology

Background:

  • Kinetic proofreading is a natural mechanism that enhances molecular recognition specificity by utilizing chemical energy to create non-equilibrium conditions.
  • This process allows for the repeated exploitation of small free-energy differences between correct and incorrect molecular targets.
  • Despite its biological significance, kinetic proofreading has been underexplored in synthetic applications like nucleic acid nanotechnology.

Purpose of the Study:

  • To introduce a novel DNA strand displacement-based kinetic proofreading motif for synthetic molecular recognition.
  • To demonstrate the enhancement of molecular recognition during templated dimerization reactions using this DNA-based fuel consumption.
  • To evaluate the ability of kinetic proofreading to improve probe specificity in discriminating single nucleotide mutations.

Main Methods:

  • Development of a DNA strand displacement system to implement kinetic proofreading.
  • Utilizing a DNA-based fuel to drive the recognition interaction out of equilibrium.
  • Employing a templated dimerization reaction to assess molecular recognition enhancement.
  • Testing probe specificity against single nucleotide mutations under kinetic proofreading conditions.

Main Results:

  • The DNA strand displacement motif successfully implemented kinetic proofreading.
  • Consumption of DNA fuel enhanced molecular recognition in the templated dimerization reaction.
  • Kinetic proofreading significantly improved probe specificity for single nucleotide mutations.
  • Enhanced specificity was observed in both the initial reaction rates and long-time behaviors of the probe.

Conclusions:

  • A DNA strand displacement-based kinetic proofreading system was successfully developed and demonstrated.
  • This synthetic kinetic proofreading approach enhances molecular recognition and specificity in DNA-based systems.
  • The findings have implications for advancing nucleic acid nanotechnology and molecular diagnostics by improving discrimination of genetic variations.