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Before a cell can divide, it must accurately replicate all of its chromosomes, including the DNA and its associated histone and non-histone proteins.  This process begins at numerous origins of replication during the S phase of the cell cycle in each of a cell’s chromosomes simultaneously. Certain nucleotides can act as origins of replication, but these sequences are not well defined - especially in complex, multi-cellular, eukaryotic species. The length of DNA that spans an origin...
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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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Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates.

Christoph F Kurat1, Joseph T P Yeeles1, Harshil Patel2

  • 1Clare Hall Laboratory, Francis Crick Institute, South Mimms, Hertfordshire EN6 3LD, UK.

Molecular Cell
|December 20, 2016
PubMed
Summary

This study reconstitutes eukaryotic DNA replication in vitro, revealing how chromatin ensures replication origin specificity and requires factors like FACT for replisome progression. It also shows how chromatin aids lagging-strand synthesis and nucleosome reassembly.

Keywords:
DNA replicationbiochemistrychromatin

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Eukaryotic genome integrity relies on efficient and controlled chromatin replication.
  • The precise mechanisms governing chromatin replication remain largely unknown.

Purpose of the Study:

  • To reconstitute eukaryotic DNA replication in vitro using purified yeast proteins and chromatinized templates.
  • To elucidate the role of chromatin in regulating DNA replication origin specificity and replisome progression.

Main Methods:

  • Reconstitution of DNA replication in vitro using purified yeast replication proteins.
  • Utilizing fully chromatinized DNA templates for experimental assays.
  • Investigating the function of specific chromatin factors, including FACT, Nhp6, INO80, ISW1A, Gcn5, and Esa1.

Main Results:

  • Chromatin enforces DNA replication origin specificity by inhibiting non-specific MCM helicase loading.
  • Histone chaperone FACT is essential for replisome progression on chromatin.
  • Nucleosome remodelers and acetyltransferases individually enhance DNA synthesis rates.
  • Chromatin facilitates lagging-strand DNA synthesis priming and efficient nucleosome reassembly on nascent DNA.

Conclusions:

  • This study defines the minimal requirements for in vitro chromatin replication.
  • Multiple chromatin factors modulate replication fork rates and ensure genome stability in vivo.