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Related Concept Videos

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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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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Fixing Double-strand Breaks02:04

Fixing Double-strand Breaks

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The double-stranded structure of DNA has two major advantages. First, it serves as a safe repository of genetic information where one strand serves as the back-up in case the other strand is damaged. Second, the double-helical structure can be wrapped around proteins called histones to form nucleosomes, which can then be tightly wound to form chromosomes. This way, DNA chains up to 2 inches long can be contained within microscopic structures in a cell. A double-stranded break not only damages...
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Conservative Site-specific Recombination and Phase Variation02:53

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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Long-patch Base Excision Repair01:02

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Since the discovery of the two BER pathways, there has been a debate about how a cell chooses one pathway over the other and the factors determining this selection. Numerous in vitro experiments have pointed out multiple determinants for the sub-pathway selection. These are:
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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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Related Experiment Video

Updated: Jun 5, 2025

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae
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Two-ended recombination at a Flp-nickase-broken replication fork.

Rajula Elango1, Namrata M Nilavar1, Andrew G Li1

  • 1Department of Medicine, Division of Hematology-Oncology and Cancer Research Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA.

Molecular Cell
|December 4, 2024
PubMed
Summary
This summary is machine-generated.

Replication fork collision with DNA nicks can cause instability. Our study shows a single fork collision with a nick triggers two-ended homologous recombination (HR) repair, potentially limiting genomic instability.

Keywords:
BRCA1CamptothecinDNA nickDNA-protein crosslinkFlp recombinaseTus/Terbreak-induced replicationhomologous recombinationreplication fork breakagereplication stress

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Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy
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Area of Science:

  • Molecular Biology
  • Genetics
  • DNA Repair

Background:

  • DNA nicks pose a risk to genomic stability, especially during replication.
  • Replication fork collision with nicks can lead to one-ended breaks, but the opposing fork might create a second end for repair.
  • Homologous recombination (HR) is a key pathway for repairing DNA breaks.

Purpose of the Study:

  • To investigate the mechanisms of nickase-induced HR in mammalian cells.
  • To characterize the pathways involved in repairing nicks encountered by replication forks.
  • To determine the origin of the second DNA end during nickase-induced HR.

Main Methods:

  • Development of a Flp recombinase "step arrest" nickase in mammalian cells.
  • Analysis of homologous recombination pathways, including BRCA2/RAD51-dependent and independent gene conversion.
  • Utilizing a Tus/Ter replication fork barrier (RFB) to block opposing forks.

Main Results:

  • Flp-nickase induces both short tract gene conversion (STGC) and long tract gene conversion (LTGC).
  • HR pathways induced by Flp-nickase differ in their dependence on BRCA1, MRE11, and CtIP compared to replication-independent breaks.
  • Blocking the opposing fork with Tus/Ter RFB did not abolish two-ended STGC, indicating a robust response.

Conclusions:

  • A single replication fork's collision with a Flp-nick triggers two-ended HR.
  • This response may involve replicative bypass of lagging strand nicks.
  • This mechanism potentially limits genomic instability arising from replication of nicked DNA.