Complex Chromosomal Rearrangements Mediated by Break-Induced Replication Involve Structure-Selective Endonucleases Benjamin Pardo, Andre´ s Aguilera* Centro Andaluz de Biolog´ıa Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla, Sevilla, Spain Abstract DNA double-strand break (DSB) repair occurring in repeated DNA sequences often leads to the generation of chromosomal rearrangements. Homologous recombination normally ensures a faithful repair of DSBs through a mechanism that transfers the genetic information of an intact donor template to the broken molecule. When only one DSB end shares homology to the donor template, conventional gene conversion fails to occur and repair can be channeled to a recombination- dependent replication pathway termed break-induced replication (BIR), which is prone to produce chromosome non- reciprocal translocations (NRTs), a classical feature of numerous human cancers. Using a newly designed substrate for the analysis of DSB–induced chromosomal translocations, we show that Mus81 and Yen1 structure-selective endonucleases (SSEs) promote BIR, thus causing NRTs. We propose that Mus81 and Yen1 are recruited at the strand invasion intermediate to allow the establishment of a replication fork, which is required to complete BIR. Replication template switching during BIR, a feature of this pathway, engenders complex chromosomal rearrangements when using repeated DNA sequences dispersed over the genome. We demonstrate here that Mus81 and Yen1, together with Slx4, also promote template switching during BIR. Altogether, our study provides evidence for a role of SSEs at multiple steps during BIR, thus participating in the destabilization of the genome by generating complex chromosomal rearrangements. Citation: Pardo B, Aguilera A (2012) Complex Chromosomal Rearrangements Mediated by Break-Induced Replication Involve Structure-Selective Endonucleases. PLoS Genet 8(9): e1002979. doi:10.1371/journal.pgen.1002979 Editor: Michael Lichten, National Cancer Institute, United States of America Received October 26, 2011; Accepted August 8, 2012; Published September 27, 2012 Copyright: 2012 Pardo, Aguilera. 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 funded by grants from the Spanish Ministry of Science and Innovation (BFU2006-05260, BFU2010-16370, and Consolider Ingenio 2010 CSD2007-015), Junta de Andaluc´ıa (BIO-102 and CVI-4567), and the European Union (FEDER). BP was supported by fellowships from Fondation Recherche Me´dicale (SPE20061209019) and EMBO (1003-2006) and a contract of the Juan de la Cierva Program of the Spanish Ministry of Science and Innovation (JCI 2009-04101). 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: aguilo@us.es Introduction The maintenance of genome integrity is crucial to prevent cell death in all organisms. Chromosomal rearrangements such as reciprocal translocations, deletions, inversions and duplications threaten genomic stability and must be avoided to prevent cancer development and genomic disorders [1]. The occurrence of unfaithful repair of DNA double-strand breaks (DSBs) is widely admitted to be the main source of chromosomal rearrangements [2–5]. Nonhomologous End Joining (NHEJ) and Homologous recombination (HR) constitute the main pathways of DSB repair. While NHEJ seal the broken DNA ends by simple religation, HR uses sequence homology between the DSB ends and an intact template for repair and is typically considered as error free. Nevertheless, in a number of cases, such as when HR occurs between non-allelic DNA sequences or DNA repeated sequences or HR is used for the repair of DSB ends containing different levels of similarity, irreversible genomic changes can take place. Thus, when only one end of a DSB shares homology with other sequences in the genome, repair by HR can occur through a replication mechanism termed break-induced replication (BIR) that often gives rise to non-reciprocal translocations (NRTs) [6–9]. BIR requires HR canonical factors such as Rad52 and Rad51 to allow efficient strand invasion of the repair template and to form a Displacement-loop (D-loop) that can be extended by DNA synthesis from the invading 39 DSB end [10,11]. In the absence of another DSB end to capture the newly synthesized strand or to independently invade the homologous template, the strand invasion intermediate is thought to be converted into a DNA replication fork capable of replicating an entire chromosome arm until encountering a telomere, a centromere, or a converging replication fork [12–15]. A key factor in this process is Pol32, the non-essential subunit of DNA polymerase d, which is dispensable for normal replication but essential for BIR [13]. Another feature of BIR is the unstable nature of its replication intermediates. It has been shown that DSB repair by BIR can occur through several rounds of strand invasion, synthesis, and dissociation from the invaded template [16]. Within dispersed repeated sequences, template switching during BIR can generate complex chromo- somal rearrangements [16–21]. BIR reactions can also be aborted to end in half-crossovers [22,23]. Half-crossovers cause NRTs, leaving the template that has been used for repair broken. They are similar to the NRTs observed in humans, which are involved in the cascade of genomic instability characteristic of human cancer cells [24]. Little is known about how BIR intermediates are processed to allow the establishment of a replication fork after strand invasion, and to cause template switching and half-crossovers. Eukaryotic PLOS Genetics | www.plosgenetics.org 1 September 2012 | Volume 8 | Issue 9 | e1002979