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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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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.
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DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart,...
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DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis
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Engineering Ca2+-Dependent DNA Polymerase Activity.

Bradley W Biggs1,2, Alexandra M de Paz3,2, Namita J Bhan1,2

  • 1Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States.

ACS Synthetic Biology
|October 19, 2023
PubMed
Summary
This summary is machine-generated.

Researchers engineered DNA polymerases (DNAPs) to detect calcium ions (Ca2+), overcoming slow signal transduction in DNA-based biosensors. This breakthrough enables faster, more responsive biosensing for diverse applications.

Keywords:
biosensorsdigital DNAprotein engineering

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Area of Science:

  • Synthetic biology
  • Biosensing technologies
  • Molecular engineering

Background:

  • Synthetic biology advances biosensing for diagnostics and genetic programming.
  • Next-generation biosensors require expanded measurement environments, phenomena, and improved quality.
  • DNA integration in biosensors offers data recording but faces signal transduction bottlenecks.

Purpose of the Study:

  • To engineer DNA polymerases (DNAPs) for sensing and responding to calcium ions (Ca2+).
  • To overcome signal transduction limitations in DNA-based biosensor recording.
  • To establish a foundation for developing novel DNAP-based biosensors.

Main Methods:

  • Engineering components of the Pol δ replicative polymerase complex from *Saccharomyces cerevisiae*.
  • Utilizing domain insertion and binding site grafting onto Pol δ subunits.
  • Demonstrating functional allosteric sensitivity to Ca2+.

Main Results:

  • Successfully engineered DNAPs with allosteric sensitivity to Ca2+.
  • Demonstrated Ca2+ responsiveness in modified Pol δ subunits.
  • Established a novel approach for ligand-gated DNAP activity.

Conclusions:

  • Engineered DNAPs can function as both sensors and responders to specific ligands like Ca2+.
  • This work addresses the signal transduction bottleneck in DNA-based biosensors.
  • Provides a foundation for developing advanced DNAP-based biosensing platforms.