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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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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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Replication in Prokaryotes01:32

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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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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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Visualizing Single-molecule DNA Replication with Fluorescence Microscopy
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Measuring DNA Replication in Hypoxic Conditions.

Iosifina P Foskolou1, Deborah Biasoli1, Monica M Olcina1

  • 1Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Oxford, OX3 7DQ, UK.

Advances in Experimental Medicine and Biology
|June 22, 2016
PubMed
Summary
This summary is machine-generated.

Maintaining replication fork integrity is crucial for preventing DNA damage and cell death. This study adapts the DNA fiber assay for studying DNA replication under hypoxia, a common tumor condition.

Keywords:
DNA fiber assayDNA replicationHypoxia

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Cancer Research

Background:

  • Cell division requires intact replication forks to prevent DNA damage and cell death.
  • Replication alterations can lead to genomic instability and cancer.
  • The DNA fiber assay is a sensitive method for studying DNA replication.

Purpose of the Study:

  • To adapt the DNA fiber assay for studying DNA replication under hypoxic conditions.
  • To provide a method for overcoming technical challenges associated with hypoxic chambers.

Main Methods:

  • Sequential pulse labeling of live cells with two thymidine analogues.
  • Detection using specific antibodies and fluorescence imaging.
  • Performing the DNA fiber assay within hypoxic chambers.

Main Results:

  • Direct examination of DNA synthesis sites at the single molecule level.
  • Quantitative and qualitative analysis of DNA replication under hypoxia.
  • Identification of strategies to manage technical issues in hypoxic assays.

Conclusions:

  • The adapted DNA fiber assay enables the study of DNA replication under physiologically relevant hypoxic stress.
  • This method is valuable for understanding genomic instability in solid tumors.
  • The described adaptations facilitate research in tumor microenvironments.