A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin

Aniek Janssen; Gregory A. Breuer; Eva K. Brinkman; Annelot I. van der Meulen; Sean V. Borden; Bas van Steensel; Ranjit S. Bindra; Jeannine R. LaRocque; Gary H. Karpen

doi:10.1101/gad.283028.116

A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin

¹Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA;
²Department of Therapeutic Radiology, Yale School of Medicine, New Haven, Connecticut 06510, USA;
³Department of Experimental Pathology, Yale School of Medicine, New Haven, Connecticut 06510, USA;
⁴Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam 1066 CX, the Netherlands;
⁵Department of Human Science, School of Nursing and Health Studies, Georgetown University Medical Center, Washington, DC 20057, USA;
⁶Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA

Corresponding authors: ghkarpen{at}lbl.gov, jan.larocque{at}georgetown.edu

Abstract

Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context.

Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here, we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster. Live imaging of single DSBs in larval imaginal discs recapitulates the spatio–temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains.

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Footnotes

Supplemental material is available for this article.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.283028.116.

Received April 21, 2016.
Accepted July 5, 2016.

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