The Role of DNA Double-Strand Breaks in Spontaneous Homologous Recombination in S. cerevisiae

Abstract

Homologous recombination (HR) is a source of genomic instability and the loss of heterozygosity in mitotic cells. Since these events pose a severe health risk, it is important to understand the molecular events that cause spontaneous HR. In eukaryotes, high levels of HR are a normal feature of meiosis and result from the induction of a large number of DNA double-strand breaks (DSBs). By analogy, it is generally believed that the rare spontaneous mitotic HR events are due to repair of DNA DSBs that accidentally occur during mitotic growth. Here we provide the first direct evidence that most spontaneous mitotic HR in Saccharomyces cerevisiae is initiated by DNA lesions other than DSBs. Specifically, we describe a class of rad52 mutants that are fully proficient in inter- and intra-chromosomal mitotic HR, yet at the same time fail to repair DNA DSBs. The conclusions are drawn from genetic analyses, evaluation of the consequences of DSB repair failure at the DNA level, and examination of the cellular re-localization of Rad51 and mutant Rad52 proteins after introduction of specific DSBs. In further support of our conclusions, we show that, as in wild-type strains, UV-irradiation induces HR in these rad52 mutants, supporting the view that DNA nicks and single-stranded gaps, rather than DSBs, are major sources of spontaneous HR in mitotic yeast cells. The genome of any organism is constantly damaged as an inevitable result of its own metabolism and exposure to irradiation. For that reason all organisms have developed many DNA repair systems to cope with the different types of DNA damage that challenge the stability of their genomes during daily life. One of these repair mechanisms is based on homologous recombination (HR), which, as a side effect, may result in loss of heterozygosity. For example, if a diploid organism harbors one functional and one dysfunctional copy of an important gene, DNA repair by HR may lead to a cell where both copies are defective. Since loss of heterozygosity plays a major role in tumorigenesis in higher eukaryotes, it is important to understand what types of DNA damage trigger HR most efficiently. In this paper, the authors have used a yeast-based system to investigate this topic, and based on mutations that separate the functions of Rad52 (a protein that is essential for HR) they conclude that DNA double-strand breaks are not the lesions that initiate most HR, but rather it is due to DNA nicks and single-stranded DNA regions.