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Related Experiment Videos

Break-induced replication: a review and an example in budding yeast.

E Kraus1, W Y Leung, J E Haber

  • 1Department of Biology and Rosenstiel Center, Brandeis University, Waltham, MA 02454-9110, USA.

Proceedings of the National Academy of Sciences of the United States of America
|July 19, 2001
PubMed
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Break-induced replication (BIR) repairs broken chromosomes by restarting DNA replication and maintaining telomeres. This study shows BIR can drive gene targeting in yeast, involving new DNA synthesis and nonhomologous end-joining.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Break-induced replication (BIR) is crucial for repairing broken chromosomes and maintaining genome integrity.
  • BIR restarts DNA replication at broken forks and preserves telomeres when telomerase is absent.
  • Gene targeting mechanisms were previously thought to involve simple crossovers, but extensive DNA synthesis resembling BIR was suggested.

Purpose of the Study:

  • To investigate the role of BIR in gene targeting in Saccharomyces cerevisiae.
  • To examine how DNA fragments with limited homology initiate new DNA synthesis and recombination.
  • To elucidate the mechanisms underlying gene targeting events involving extensive DNA synthesis.

Main Methods:

  • Transformation of Saccharomyces cerevisiae with linearized plasmid DNA containing specific gene homology.

Related Experiment Videos

  • Utilizing strains with modified gene sequences (LEU2, ADE1, ADE2, URA3) to prevent simple homologous gene replacement.
  • Analysis of transformants to identify gene integration, DNA synthesis, and circular DNA formation.

    • Main Results:

    • Gene targeting occurred via BIR, resulting in the integration of the entire LEU2 gene and up to 7 kb of additional sequences, joined by microhomologies characteristic of nonhomologous end-joining (NHEJ).
    • Transformed DNA fragments lacking an ARS initiated new DNA synthesis by BIR, leading to the recovery of autonomously replicating circles containing copied chromosomal sequences.
    • Extensive new DNA synthesis, characteristic of BIR, was observed during gene targeting events.

    Conclusions:

    • The end of a linearized DNA fragment can initiate new DNA synthesis through BIR.
    • Newly synthesized DNA during BIR can be displaced and subsequently form circles via NHEJ.
    • BIR is a significant mechanism in gene targeting, involving extensive DNA synthesis and recombination pathways.