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Related Concept Videos

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.
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Replication in Eukaryotes02:31

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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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During replication, the complementary strands in double-stranded DNA are synthesized at different rates. Replication first begins on the leading strand. Replication starts later, occurs more slowly, and proceeds discontinuously on the lagging strand.
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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 vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication.

Grant Schauer1, Jeff Finkelstein1, Mike O'Donnell1

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|October 31, 2017
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Summary

This study details a practical in vitro method for distinguishing leading and lagging DNA strand synthesis. It provides a guide for researchers studying DNA replication using purified proteins.

Keywords:
CMG helicaseDNA polymeraseEukaryotic DNA replicationLagging strandLeading strandPCNA sliding clampRFC clamp loaderReplisome assay

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • The eukaryotic replisome is a complex molecular machine responsible for DNA duplication.
  • It coordinates continuous leading strand synthesis and discontinuous lagging strand synthesis.
  • DNA polymerases ε (epsilon) and δ (delta) are key enzymes in this process, alongside other proteins like helicases and clamp loaders.

Purpose of the Study:

  • To provide a practical guide for researchers to assay leading and lagging strand DNA replication in vitro.
  • To enable differential analysis of these two distinct DNA synthesis processes using purified components.

Main Methods:

  • Development of an in vitro assay system.
  • Utilizing purified proteins involved in DNA replication.
  • Differential measurement techniques to distinguish leading and lagging strand synthesis.

Main Results:

  • A robust protocol for separating and analyzing leading and lagging strand synthesis was established.
  • The method allows for the study of polymerase activity and fidelity on both DNA strands.

Conclusions:

  • This practical guide facilitates detailed mechanistic studies of DNA replication forks.
  • Enables further investigation into the coordination and regulation of leading and lagging strand synthesis.