Replicative Age Induces Mitotic Recombination in the Ribosomal RNA Gene Cluster of Saccharomyces cerevisiae Derek L. Lindstrom, Christina K. Leverich, Kiersten A. Henderson, Daniel E. Gottschling* Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America Abstract Somatic mutations contribute to the development of age-associated disease. In earlier work, we found that, at high frequency, aging Saccharomyces cerevisiae diploid cells produce daughters without mitochondrial DNA, leading to loss of respiration competence and increased loss of heterozygosity (LOH) in the nuclear genome. Here we used the recently developed Mother Enrichment Program to ask whether aging cells that maintain the ability to produce respiration- competent daughters also experience increased genomic instability. We discovered that this population exhibits a distinct genomic instability phenotype that primarily affects the repeated ribosomal RNA gene array (rDNA array). As diploid cells passed their median replicative life span, recombination rates between rDNA arrays on homologous chromosomes progressively increased, resulting in mutational events that generated LOH at .300 contiguous open reading frames on the right arm of chromosome XII. We show that, while these recombination events were dependent on the replication fork block protein Fob1, the aging process that underlies this phenotype is Fob1-independent. Furthermore, we provide evidence that this aging process is not driven by mechanisms that modulate rDNA recombination in young cells, including loss of cohesion within the rDNA array or loss of Sir2 function. Instead, we suggest that the age-associated increase in rDNA recombination is a response to increasing DNA replication stress generated in aging cells. Citation: Lindstrom DL, Leverich CK, Henderson KA, Gottschling DE (2011) Replicative Age Induces Mitotic Recombination in the Ribosomal RNA Gene Cluster of Saccharomyces cerevisiae. PLoS Genet 7(3): e1002015. doi:10.1371/journal.pgen.1002015 Editor: Jasper Rine, University of California Berkeley, United States of America Received November 24, 2010; Accepted January 13, 2011; Published March 17, 2011 Copyright: 2011 Lindstrom et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by postdoctoral fellowship #PF-04-041-01-GMC from the American Cancer Society (http://www.cancer.org) to DLL, a Fellow’s Career Development Program Award from The Leukemia and Lymphoma Society (http://www.leukemia-lymphoma.org/hm_lls) to KAH, National Institutes of Health (http://www.nih.gov/) grant AG023779 and a Glenn Award for Research in Biological Mechanisms of Aging (http://glennfoundation.org/) to DEG, and National Cancer Institute (http://www.cancer.gov/) grant T32 CA09657. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dgottsch@fhcrc.org Introduction One of the greatest risk factors associated with carcinogenesis is age. Cancer risk increases exponentially toward the end of life in humans and other mammalian species [1]. Somatic genetic changes contribute significantly to the development of most tumors. However, the rate at which spontaneous mutations arise in normal adult cells has been hypothesized to be too low to generate all the genetic changes necessary to produce tumors at the observed rates [2–3]. Consequently, Loeb et al. developed the mutator hypothesis, which postulates an increased mutation rate in precancerous cells [3]. A variety of mechanisms could lead to such an increase, but a favored model is that sporadic mutations in, or epigenetic silencing of, genes responsible for maintaining genome integrity lead to increased rates of mutation. Once acquired, this mutator phenotype may serve as the driving force toward carcinogenesis as individuals age. When genomes of tumors are examined, loss of heterozygosity (LOH) is observed as a common mechanism in which the sole functional allele of a tumor suppressor gene is inactivated by somatic mutation [4–5]. LOH can be generated by many different mutational events, including point mutations, small deletions or inversions, mitotic recombination or chromosome loss. Recent advances in high- resolution single-nucleotide polymorphism arrays (SNP arrays) have revealed that a surprising number of tumors contain long tracts of homozygosity that are not accompanied by a change in gene copy number. This type of LOH arises from somatic mitotic recombina- tion events and is referred to as partial (or acquired) uniparental disomy (UPD) [6]. Importantly, UPD can alter the genotype for hundreds of genes following a single event, thereby amplifying its potential to contribute to cancer development [7]. Within any genome, there are regions that exhibit higher rates of mitotic recombination than the genomic average as the result of their proximity to hotspots or common fragile sites [8]. These regions typically represent slow-replicating sequences, and agents that generate DNA replication stress often reveal their fragility. These regions are also often associated with non-histone protein complexes that inhibit DNA replication fork progression. This generates DNA replication stress that may lead to an increased frequency of DNA damage due to replication fork collapse [9–10]. While conservative repair of this DNA damage would have no genetic consequence, the higher incidence of damage results in a greater chance that an alternative repair pathway will be utilized that does confer a genotypic change. Previously, we found evidence for a mutator phenotype associated with advancing replicative age in a common lab strain PLoS Genetics | www.plosgenetics.org 1 March 2011 | Volume 7 | Issue 3 | e1002015