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Related Concept Videos

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.
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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 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 Topoisomerases02:02

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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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The DNA Replication Fork01:02

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An organism’s genome needs to be duplicated in an efficient and error-free manner for its growth and survival. The replication fork is a Y-shaped active region where two strands of DNA are separated and replicated continuously. The coupling of DNA unzipping and complementary strand synthesis is a characteristic feature of a replication fork.   Organisms with small circular DNA, such as E. coli, often have a single origin of replication; therefore, they have only two replication...
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Updated: Dec 21, 2025

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins
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Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Published on: February 25, 2017

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Catalytically inactive T7 DNA polymerase imposes a lethal replication roadblock.

Alfredo J Hernandez1, Seung-Joo Lee1, Seungwoo Chang1

  • 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.

The Journal of Biological Chemistry
|May 21, 2020
PubMed
Summary
This summary is machine-generated.

Inactive bacteriophage T7 DNA polymerase variants are toxic to E. coli cells when complexed with thioredoxin. This interaction inhibits DNA synthesis, suggesting a potential bacterial cell-growth control mechanism.

Keywords:
DNA polymeraseDNA replicationbacterial geneticsbacteriophageenzyme mutationgene 5 (gp5)processivityreplisomethioredoxin

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Area of Science:

  • Molecular Biology
  • Virology
  • Biochemistry

Background:

  • Bacteriophage T7 DNA polymerase (gp5) has low processivity alone.
  • Complex formation with Escherichia coli thioredoxin (TrxA) significantly enhances gp5 processivity.

Purpose of the Study:

  • To investigate the biological activity and cellular effects of a gp5 variant with mutations in its metal-binding site.
  • To explore the potential of engineered gp5 variants as a bacterial cell-growth control system.

Main Methods:

  • Expression of a mutated gp5 variant in E. coli.
  • Assessment of cellular toxicity and dependence on E. coli trxA and T7 RNA polymerase.
  • In vitro characterization of the gp5 variant's polymerase activity and DNA synthesis inhibition.
  • Comparison with a modified Klenow fragment.

Main Results:

  • The gp5 variant, lacking polymerase activity, induced high toxicity in E. coli cells.
  • Toxicity was dependent on functional E. coli trxA and T7 RNA polymerase, but not gp5 exonuclease activity.
  • In vitro, the variant inhibited DNA synthesis by forming stable, replication-blocking complexes with DNA in the presence of thioredoxin.
  • A homologous Klenow fragment with a gp5 thioredoxin-binding domain did not cause toxicity.

Conclusions:

  • Inactive gp5 polymerases complexed with thioredoxin can inhibit DNA replication and cause cellular toxicity.
  • These findings suggest that specific gp5 alleles could serve as a controllable shutoff mechanism for bacterial growth systems.