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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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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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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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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 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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Mechanism for inverted-repeat recombination induced by a replication fork barrier.

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Replication stress triggers a unique recombination pathway involving Rad51 and other factors, leading to chromosome rearrangements like inversions. This study elucidates mechanisms of genomic instability in yeast.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Genomic instability in eukaryotes is often caused by replication stress and repetitive DNA sequences.
  • Understanding recombination mechanisms under replication stress is crucial for genomic stability research.

Purpose of the Study:

  • To investigate the mechanism of recombination between repeated sequences during replication stress.
  • To elucidate the factors and pathways involved in generating chromosome rearrangements.

Main Methods:

  • Utilized a prokaryotic Tus/Ter system in budding yeast to induce replication fork stalling near inverted repeats.
  • Performed extensive genetic characterization of recombination pathways and associated proteins.
  • Analyzed replication-associated recombinants to identify sequence alterations.

Main Results:

  • Replication fork stalling activates a novel recombination pathway requiring Rad51, Rad52-Rad59, Mph1/Rad5, Mre11/Exo1/Dna2, Rad1-Rad10, and DNA polymerase δ.
  • Recombination at stalled forks is regulated by Srs2 helicase and Mus81-Mms4/Yen1 nucleases.
  • Half of the identified recombinants exhibited sequence inversions between repeats.

Conclusions:

  • Propose a model for recombination of closely linked repeats that generates chromosome rearrangements under replication stress.
  • The findings provide insights into the generation of genomic instability and potential therapeutic targets.