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DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
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DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...
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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 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.
Many Proteins Work Together to Replicate the Chromosome
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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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Related Experiment Video

Updated: Sep 22, 2025

Author Spotlight: Optimizing Affinity Chromatography for His-Tagged FEN1 Protein
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Author Spotlight: Optimizing Affinity Chromatography for His-Tagged FEN1 Protein

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Fast and efficient DNA replication with purified human proteins.

Yasemin Baris1, Martin R G Taylor1, Valentina Aria1

  • 1MRC Laboratory of Molecular Biology, Cambridge, UK.

Nature
|May 18, 2022
PubMed
Summary
This summary is machine-generated.

Researchers reconstituted the human replisome, revealing DNA polymerase epsilon (Polε) is key for leading-strand synthesis. This process requires PCNA and CTF18-RFC, with AND-1 directly aiding leading-strand replication.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • The human replisome, essential for DNA replication, comprises numerous proteins organized around the CMG helicase.
  • A complete reconstitution of the human replisome from purified proteins has been lacking, hindering understanding of its precise functions.

Purpose of the Study:

  • To biochemically reconstitute functional human replisomes using purified components.
  • To elucidate the specific roles of key proteins, including DNA polymerases and accessory factors, in human DNA replication.

Main Methods:

  • Biochemical reconstitution of human replisomes using 11 purified human replication factors (43 polypeptides).
  • Assays to measure DNA replication speed and efficiency.
  • Investigating the roles of PCNA, CTF18-RFC, CLASPIN, TIMELESS-TIPIN, and AND-1 in replisome function.

Main Results:

  • Successfully reconstituted human replisomes capable of fast and efficient DNA replication.
  • Identified DNA polymerase epsilon (Polε) as crucial for optimal leading-strand synthesis, dependent on PCNA and CTF18-RFC.
  • Demonstrated that AND-1 directly enhances leading-strand replication, unlike in yeast, and Polα is recruited independently for lagging-strand priming.

Conclusions:

  • The reconstituted human replisome provides a system to study DNA replication mechanisms.
  • Revealed distinct roles for Polε, PCNA, CTF18-RFC, and AND-1 in human leading-strand synthesis.
  • Clarified the independent recruitment of Polα for lagging-strand priming in human DNA replication.