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Related Concept Videos

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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In eukaryotic cells, DNA replication is highly conserved and tightly regulated. Multiple linear chromosomes must be duplicated with high fidelity before cell division, so there are many proteins that fulfill specialized roles in the replication process. Replication occurs in three phases: initiation, elongation, and termination, and ends with two complete sets of chromosomes in the nucleus.
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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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Topoisomerases are enzymes that relax overwound DNA molecules during various cell processes, including DNA replication and transcription. These enzymes regulate positive and negative DNA supercoiling without changing the nucleotide sequence. DNA overwinding in a clockwise direction results in positively supercoiled DNA, whereas underwinding in a counterclockwise direction produces negatively supercoiled DNA.
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Updated: Jul 18, 2025

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis
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Evolution of Organic Solvent-Resistant DNA Polymerases.

Mohammed Elias1, Xiangying Guan1, Devin Hudson1

  • 1Chakrabarti Advanced Technology, LLC, PMC Group Building, 1288 Route 73, Suite 110, Mount Laurel, New Jersey 08054, United States.

ACS Synthetic Biology
|August 23, 2023
PubMed
Summary

Researchers engineered Taq polymerase for enhanced stability and activity in organic solvents, overcoming limitations in amplifying GC-rich DNA. This breakthrough reduces sequence bias in applications like sequencing and synthetic biology.

Keywords:
DNA polymerasescomputational biophysicsdirected evolutionenzyme engineeringnext-generation sequencingnonaqueous enzymology

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Enzyme Engineering

Background:

  • Thermostable polymerases revolutionized PCR, but amplifying GC-rich genes remains challenging.
  • Organic cosolvents can improve DNA polymerization by destabilizing duplexes, yet natural polymerases lack solvent tolerance.

Purpose of the Study:

  • To evolve Taq polymerase for enhanced stability and activity in organic cosolvents.
  • To overcome amplification biases associated with GC-rich DNA sequences.

Main Methods:

  • Ultrahigh-throughput droplet-based selection and deep sequencing.
  • Computational free-energy and binding affinity calculations.
  • Directed evolution of Taq polymerase.

Main Results:

  • Engineered polymerases show increased stability and activity in organic cosolvents like 1,4-butanediol, sulfolane, and 2-pyrrolidone.
  • Successful amplification of highly GC-rich and previously recalcitrant DNA templates.
  • Significantly reduced GC bias in quantitative PCR amplification.

Conclusions:

  • Developed organic solvent-resistant polymerases expand compatible solvent systems for nucleic acid polymerization.
  • These enzymes enable reduced sequence bias, crucial for applications in sequencing and synthetic biology.
  • Offers a novel approach to enhance PCR efficiency beyond thermal stability alone.