DNA dynamics during early double-strand break processing revealed by non-intrusive imaging of living cells

PLoS Genet. 2014 Mar 13;10(3):e1004187. doi: 10.1371/journal.pgen.1004187. eCollection 2014 Mar.

Abstract

Chromosome breakage is a major threat to genome integrity. The most accurate way to repair DNA double strand breaks (DSB) is homologous recombination (HR) with an intact copy of the broken locus. Mobility of the broken DNA has been seen to increase during the search for a donor copy. Observing chromosome dynamics during the earlier steps of HR, mainly the resection from DSB ends that generates recombinogenic single strands, requires a visualization system that does not interfere with the process, and is small relative to the few kilobases of DNA that undergo processing. Current visualization tools, based on binding of fluorescent repressor proteins to arrays of specific binding sites, have the major drawback that highly-repeated DNA and lengthy stretches of strongly bound protein can obstruct chromatin function. We have developed a new, non-intrusive method which uses protein oligomerization rather than operator multiplicity to form visible foci. By applying it to HO cleavage of the MAT locus on Saccharomyces cerevisiae chromosome III, we provide the first real-time analysis of resection in single living cells. Monitoring the dynamics of a chromatin locus next to a DSB revealed transient confinement of the damaged chromatin region during the very early steps of resection, consistent with the need to keep DNA ends in contact. Resection in a yku70 mutant began ∼ 10 min earlier than in wild type, defining this as the period of commitment to homology-dependent repair. Beyond the insights into the dynamics and mechanism of resection, our new DNA-labelling and -targeting method will be widely applicable to fine-scale analysis of genome organization, dynamics and function in normal and pathological contexts.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatin / genetics*
  • Chromosomes, Fungal / metabolism
  • DNA Breaks, Double-Stranded*
  • DNA Damage / genetics
  • DNA Repair / genetics*
  • DNA-Binding Proteins / genetics
  • Genome, Fungal
  • Homologous Recombination / genetics*
  • Saccharomyces cerevisiae

Substances

  • Chromatin
  • DNA-Binding Proteins

Grants and funding

This work was supported by the Institut Universitaire de France, the Human Frontier Science Organisation (RGP0044/2011), the Fondation Association de la Recherche contre le Cancer (ARC, fellowship to FG and SFI 2010 1201818), the Fondation pour la Recherche Médicale (FRM, fellowship to MD), the Ministère de l'Education Nationale et de la Recherche (PhD fellowship to HS) and the Agence Nationale de Recherche (ANR-06-BLAN-0280-01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.