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Related Concept Videos

The DNA Replication Fork01:02

The DNA Replication Fork

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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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Restarting Stalled Replication Forks02:37

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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 Topoisomerases02:02

DNA Topoisomerases

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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.
Types and Mechanism of action
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Homologous Recombination02:31

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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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Single-Strand DNA Binding Proteins01:03

Single-Strand DNA Binding Proteins

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For successful DNA replication, the unwinding of double-stranded DNA must be accompanied by stabilization and protection of the separated single strands of the DNA. This crucial task is performed by single-strand DNA-binding (SSB) proteins. They bind to the DNA in a sequence-independent manner, which means that the nitrogenous bases of the DNA need not be present in a specific order for binding of SSB proteins to it. The binding of SSB proteins straightens single-stranded DNA (ssDNA) and makes...
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Replication in Prokaryotes01:32

Replication in Prokaryotes

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DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the single origin of replication (ori) on the cell's circular chromosome. Replication then proceeds around the entire circle of the chromosome in each direction from the two replication forks, resulting in two DNA molecules.
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Updated: Jul 15, 2025

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System
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Replication-induced DNA secondary structures drive fork uncoupling and breakage.

Sophie L Williams1, Corella S Casas-Delucchi1, Federica Raguseo2,3

  • 1Genome Replication Lab, Division of Cancer Biology, Institute of Cancer Research, Chester Beatty Laboratories, London, UK.

The EMBO Journal
|October 2, 2023
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Summary

DNA secondary structures like G-quadruplexes (G4s) and intercalated-Motifs (iMs) can halt DNA replication. Pif1 helicase resolves these structures, preventing genome instability and replication stress.

Keywords:
DNA replicationDNA secondary structuresG-quadruplex and i-Motifgenome stabilityreplication stress

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Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase
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Area of Science:

  • Genetics
  • Molecular Biology
  • Genomics

Background:

  • Abundant DNA secondary structures, including G-quadruplexes (G4s) and intercalated-Motifs (iMs), are prevalent in the human genome.
  • These structures play physiological roles but can also impede DNA replication and compromise genome stability.
  • The precise mechanisms by which G4s and iMs interfere with replication remain incompletely understood.

Purpose of the Study:

  • To investigate how G-quadruplexes (G4s) and intercalated-Motifs (iMs) affect DNA replication.
  • To elucidate the mechanistic basis of replication arrest induced by these DNA structures.
  • To identify factors involved in the resolution of replication forks stalled by G4s and iMs.

Main Methods:

  • Reconstitution of DNA replication using physiologically relevant structure-forming sequences.
  • Single-molecule structure detection in solid-state nanopores.
  • Combined genetic and biophysical characterization of structure stability and formation probability.
  • Analysis of helicase activity in resolving stalled replication forks.

Main Results:

  • A single G4 or iM is sufficient to arrest DNA replication.
  • These structures form during replication, as detected by nanopore analysis.
  • Replication arrest results from impaired synthesis and helicase-polymerase uncoupling; iMs also cause nascent DNA breakage.
  • Only the Pif1 helicase, not Rrm3, Sgs1, Chl1, or Hrq1, can rescue stalled forks.

Conclusions:

  • G4s and iMs are endogenous sources of replication stress, directly inhibiting DNA replication.
  • Structure stability and formation probability are critical determinants of replication fork arrest.
  • Pif1 is a key helicase for resolving replication-associated G4 and iM structures.