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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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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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Overcoming a nucleosomal barrier to replication.

Han-Wen Chang1, Manjula Pandey2, Olga I Kulaeva1

  • 1Fox Chase Cancer Center, Philadelphia, PA 19111, USA.

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Summary
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DNA replication encounters nucleosomes as barriers, but T7 replisome can traverse them. A novel mechanism involving DNA looping may maintain nucleosome structure during replication.

Keywords:
Replicationchromatinmechanismnucleosome

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Area of Science:

  • Molecular Biology
  • Epigenetics
  • Biochemistry

Background:

  • Maintaining chromatin structure during DNA replication is crucial for daughter cell function.
  • The precise molecular mechanisms of replication through nucleosomes remain largely unknown.

Purpose of the Study:

  • To investigate the in vitro traversal of mononucleosomes by the T7 replisome.
  • To elucidate the molecular mechanisms governing replication through chromatin.

Main Methods:

  • Studied the traversal of uniquely positioned mononucleosomes by the T7 replisome in vitro.
  • Analyzed the impact of nucleosome position and T7 DNA polymerase exonuclease activity on replication fork progression.

Main Results:

  • Nucleosomes act as sequence-dependent barriers to replication, causing significant pausing of DNA polymerase.
  • T7 DNA polymerase's exonuclease activity enhances replisome progression through nucleosomes.
  • Nucleosome-free DNA upstream of the replication fork aids polymerase traversal.
  • Over 50% of nucleosomes maintain their original positions post-replication, adopting an alternative conformation.

Conclusions:

  • Replication through chromatin involves overcoming strong nucleosomal barriers.
  • T7 replisome utilizes exonuclease activity and benefits from upstream nucleosome-free DNA for efficient traversal.
  • A novel mechanism, potentially involving transient intranucleosomal DNA looping, is proposed for nucleosome maintenance during replication.