Break-Induced Loss of Heterozygosity in Fission Yeast: Dual Roles for Homologous Recombination in Promoting Translocations and Preventing De Novo Telomere Addition

Abstract

Loss of heterozygosity (LOH), a causal event in tumorigenesis, frequently encompasses multiple genetic loci and whole chromosome arms. However, the mechanisms leading to such extensive LOH are poorly understood. We investigated the mechanisms of DNA double-strand break (DSB)-induced extensive LOH by screening for auxotrophic marker loss ∼25 kb distal to an HO endonuclease break site within a nonessential minichromosome in Schizosaccharomyces pombe. Extensive break-induced LOH was infrequent, resulting from large translocations through both allelic crossovers and break-induced replication. These events required the homologous recombination (HR) genes rad32⁺, rad50⁺, nbs1⁺, rhp51⁺, rad22⁺, rhp55⁺, rhp54⁺, and mus81⁺. Surprisingly, LOH was still observed in HR mutants, which resulted predominantly from de novo telomere addition at the break site. De novo telomere addition was most frequently observed in rad22Δ and rhp55Δ backgrounds, which disrupt HR following end resection. Further, levels of de novo telomere addition, while increased in ku70Δ rhp55Δ strains, were reduced in exo1Δ rhp55Δ and an rhp55Δ strain overexpressing rhp51. These findings support a model in which HR prevents de novo telomere addition at DSBs by competing for resected ends. Together, these results suggest that the mechanisms of break-induced LOH may be predicted from the functional status of the HR machinery.

SUPPLEMENTAL MATERIAL

We thank the laboratories of Nick Boddy, Tony Carr, Stefania Francesconi, Chris Norbury, Paul Russell, Masaru Ueno, and Matthew Whitby for strains and reagents.

This research was funded by the Medical Research Council. B.-Y.W. was funded by the Agency for Science Technology and Research, Singapore.