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Related Concept Videos

Replication in Eukaryotes01:29

Replication in Eukaryotes

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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 Replication02:40

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DNA replication involves the separation of the two strands of the double helix, with each strand serving as a template from which the new complementary strand is copied.  After replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. This is known as semiconservative replication. The resulting DNA molecules have the same sequence and are divided equally into the two daughter cells.
Replication in Prokaryotes
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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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The cell cycle is a series of events leading to DNA duplication followed by the division of cell content to form two daughter cells. The cell cycle progresses in four stages—the cell increases in size (gap 1 or G1-phase), duplicates its DNA (synthesis or S-phase), prepares to divide (gap 2 or G2-phase), and divides (mitosis or M-phase).
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A functional eukaryotic chromosome must contain three elements: a centromere, telomeres, and numerous origins of replication.
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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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Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method
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Eukaryotic DNA replication with purified budding yeast proteins.

Viktor Posse1, Erik Johansson2, John F X Diffley3

  • 1Chromosome Replication Laboratory, The Francis Crick Institute, London, United Kingdom; Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden.

Methods in Enzymology
|November 15, 2021
PubMed
Summary

Reconstituting eukaryotic DNA replication in vitro is crucial. This study presents optimized protocols for 24 protein purifications and a reliable in vitro replication assay for budding yeast, improving efficiency and troubleshooting.

Keywords:
Chromosome duplicationDNA replicationDNA replication assayDNA replication in vitroProtein expressionProtein purificationReconstituted yeast DNA replication

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • The in vitro reconstitution of origin firing was a significant advancement for studying eukaryotic DNA replication.
  • Current replication assays rely on proteins purified through 24 distinct, evolved protocols.
  • Improvements in purification protocols have enhanced the efficiency and reliability of in vitro replication systems.

Purpose of the Study:

  • To consolidate and present all 24 established protein purification protocols for eukaryotic DNA replication in budding yeast.
  • To provide a standardized, general protocol for the in vitro replication assay.
  • To offer troubleshooting guidance for common issues encountered in the in vitro replication assay.

Main Methods:

  • Detailed protocols for the purification of 24 individual proteins involved in DNA replication.
  • A comprehensive, step-by-step protocol for the in vitro replication assay.
  • Compilation of troubleshooting tips based on practical experience.

Main Results:

  • A unified set of 24 optimized protein purification protocols is now available.
  • A reliable and efficient general protocol for the in vitro replication assay has been established.
  • The presented protocols facilitate consistent and reproducible results in DNA replication studies.

Conclusions:

  • This work provides a valuable resource for researchers studying eukaryotic DNA replication.
  • The standardized protocols enhance the accessibility and reproducibility of in vitro replication assays.
  • Improved methods contribute to a deeper understanding of DNA replication mechanisms in budding yeast.