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Double-strand break repair in yeast requires both leading and lagging strand DNA polymerases

A M Holmes1, J E Haber

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

Cell
|February 20, 1999
PubMed

Insights

DNA repair via gene conversion requires replication factors, including processivity cofactors like PCNA and RFC. Lagging strand synthesis is surprisingly crucial for completing double-strand break repair.

Area of Science:

  • Molecular biology
  • Yeast genetics
  • DNA replication and repair

Background:

  • Double-strand breaks (DSBs) are severe DNA lesions that can lead to genomic instability.
  • Gene conversion is a key mechanism for repairing DSBs, particularly in programmed processes like mating type switching in Saccharomyces cerevisiae.
  • Replication factors are known to be involved in DNA repair, but their precise roles in DSB-induced gene conversion are complex.

Purpose of the Study:

  • To investigate the roles of essential replication factors in DSB-induced gene conversion at the MAT locus in yeast.
  • To elucidate the molecular mechanisms by which replication machinery participates in the repair of mitotic DSBs.

Main Methods:

  • Utilized thermosensitive mutant strains of Saccharomyces cerevisiae defective in essential replication factors.
  • Performed molecular analysis of gene conversion events following induced DSBs.
  • Assessed the impact of mutations in processivity cofactors, DNA polymerases, and lagging strand replication machinery.

Main Results:

  • Processivity cofactors (PCNA and RFC) are essential for DNA synthesis post-strand invasion, even for short stretches.
  • Both DNA polymerases delta and epsilon are important for gene conversion, with Pol epsilon playing a more critical role.
  • Mutants affecting lagging strand synthesis (DNA polymerase alpha, DNA primase, Rad27p) significantly inhibit DSB repair completion, even in non-cycling cells.

Conclusions:

  • DSB-induced gene conversion involves a modified replication fork utilizing both leading and lagging strand synthesis from a donor template.
  • Lagging strand replication machinery plays a critical, previously underappreciated role in completing DSB repair.
  • Replication termination occurs through the capture of the second end of the DSB, integrating newly synthesized DNA.

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