The Caenorhabditis elegans Homolog of Gen1/Yen1 Resolvases Links DNA Damage Signaling to DNA Double-Strand Break Repair Aymeric P. Bailly 1 , Alasdair Freeman 2. , Julie Hall 3.¤a , Anne-Ce ´ cile De ´ clais 2. , Arno Alpi 1¤b , David M. J. Lilley 2 , Shawn Ahmed 3 , Anton Gartner 1 * 1 Wellcome Trust Centre for Gene Regulation and Expression, University of Dundee, Dundee, United Kingdom, 2 Cancer Research United Kingdom Nucleic Acid Structure Research Group, University of Dundee, Dundee, United Kingdom, 3 Department of Genetics and Department of Biology, University of North Carolina, Chapel Hill, North Carolina, United States of America Abstract DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR), which can involve Holliday junction (HJ) intermediates that are ultimately resolved by nucleolytic enzymes. An N-terminal fragment of human GEN1 has recently been shown to act as a Holliday junction resolvase, but little is known about the role of GEN-1 in vivo. Holliday junction resolution signifies the completion of DNA repair, a step that may be coupled to signaling proteins that regulate cell cycle progression in response to DNA damage. Using forward genetic approaches, we identified a Caenorhabditis elegans dual function DNA double-strand break repair and DNA damage signaling protein orthologous to the human GEN1 Holliday junction resolving enzyme. GEN-1 has biochemical activities related to the human enzyme and facilitates repair of DNA double-strand breaks, but is not essential for DNA double-strand break repair during meiotic recombination. Mutational analysis reveals that the DNA damage-signaling function of GEN-1 is separable from its role in DNA repair. GEN-1 promotes germ cell cycle arrest and apoptosis via a pathway that acts in parallel to the canonical DNA damage response pathway mediated by RPA loading, CHK1 activation, and CEP-1/p53–mediated apoptosis induction. Furthermore, GEN-1 acts redundantly with the 9-1-1 complex to ensure genome stability. Our study suggests that GEN-1 might act as a dual function Holliday junction resolvase that may coordinate DNA damage signaling with a late step in DNA double-strand break repair. Citation: Bailly AP, Freeman A, Hall J, De ´clais A-C, Alpi A, et al. (2010) The Caenorhabditis elegans Homolog of Gen1/Yen1 Resolvases Links DNA Damage Signaling to DNA Double-Strand Break Repair. PLoS Genet 6(7): e1001025. doi:10.1371/journal.pgen.1001025 Editor: Tim Schedl, Washington University, United States of America Received December 24, 2009; Accepted June 14, 2010; Published July 15, 2010 Copyright: ß 2010 Bailly 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: Funding was provided by Cancer Research UK (CDA award to AG and programme grant to DMJL), the Wellcome Trust, and SA and JH were supported by NIH grant GM066228 and by an Ellison Medical Foundation New Scholar in Aging Award. 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: a.gartner@dundee.ac.uk . These authors contributed equally to this work. ¤a Current address: Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, Durham, North Carolina, United States of America ¤b Current address: The Scottish Institute for Cell Signalling, University of Dundee, Dundee, United Kingdom Introduction The correct maintenance and duplication of genetic information is constantly challenged by genotoxic stress. DNA double-strand breaks (DSBs) are amongst the most deleterious lesions. DSBs can be induced by ionizing irradiation (IR) or caused by the stalling of DNA replication forks. In response to DSBs, cells activate conserved DNA damage checkpoint pathways that lead to DNA repair, to a transient cell cycle arrest, or to apoptosis and senescence. The full activation of DNA damage response pathways and DSB repair by homologous recombination (HR) depends on a series of nucleolytic processing events. Following DSB formation, broken ends are resected in a 59 to 39 direction to generate 39 single-strand overhangs [1]. These tails are coated by RPA1 molecules, which in turn are thought to lead to the recruitment of the ATR checkpoint kinase [2]. This kinase, and the related kinase ATM, appear to be directly targeted to DNA double-strand breaks to act at the apex of the DNA damage signaling cascade [3]. The DNA damage specific clamp loader comprised of Rad17 bound to the four smallest RFC subunits [4] recruits a PCNA-like complex referred to as ‘‘9-1-1’’ complex to the dsDNA–ssDNA transition of resected DNA ends [5–7]. The 9-1-1 complex is needed for full ATR activation [8,9]. DSB repair by HR proceeds by replacing RPA1 with the RAD51 recombinase [10,11]. The resulting nucleoprotein filament invades an intact donor DNA to form a D-loop structure. The invading strand is extended using the intact donor strand as a template. Annealing of the 39 single-stranded tail of the second resected DNA end to the displaced donor DNA strand (second end capture), and DNA ligation lead to the formation of a double Holliday junction (dHJ) intermediate (for a review, see [12]). This dHJ must be resolved either through cleavage by Holliday junction (HJ)-resolving enzymes or through ‘‘dissolution’’ by the combined activity of the Blooms helicase and topoisomerase III [13,14]. Prototypic HJ resolving enzymes are nucleases that resolve HJs by introducing two symmetrical cleavages that result in either crossover or non-crossover products, depending on which strands PLoS Genetics | www.plosgenetics.org 1 July 2010 | Volume 6 | Issue 7 | e1001025