Mre11-Rad50 Promotes Rapid Repair of DNA Damage in the Polyploid Archaeon Haloferax volcanii by Restraining Homologous Recombination Ste ´ phane Delmas, Lee Shunburne, Hien-Ping Ngo ¤ , Thorsten Allers* Institute of Genetics, School of Biology, University of Nottingham, Queen’s Medical Centre, Nottingham, United Kingdom Abstract Polyploidy is frequent in nature and is a hallmark of cancer cells, but little is known about the strategy of DNA repair in polyploid organisms. We have studied DNA repair in the polyploid archaeon Haloferax volcanii, which contains up to 20 genome copies. We have focused on the role of Mre11 and Rad50 proteins, which are found in all domains of life and which form a complex that binds to and coordinates the repair of DNA double-strand breaks (DSBs). Surprisingly, mre11 rad50 mutants are more resistant to DNA damage than the wild-type. However, wild-type cells recover faster from DNA damage, and pulsed-field gel electrophoresis shows that DNA double-strand breaks are repaired more slowly in mre11 rad50 mutants. Using a plasmid repair assay, we show that wild-type and mre11 rad50 cells use different strategies of DSB repair. In the wild-type, Mre11-Rad50 appears to prevent the repair of DSBs by homologous recombination (HR), allowing microhomology-mediated end-joining to act as the primary repair pathway. However, genetic analysis of recombination- defective radA mutants suggests that DNA repair in wild-type cells ultimately requires HR, therefore Mre11-Rad50 merely delays this mode of repair. In polyploid organisms, DSB repair by HR is potentially hazardous, since each DNA end will have multiple partners. We show that in the polyploid archaeon H. volcanii the repair of DSBs by HR is restrained by Mre11-Rad50. The unrestrained use of HR in mre11 rad50 mutants enhances cell survival but leads to slow recovery from DNA damage, presumably due to difficulties in the resolution of DNA repair intermediates. Our results suggest that recombination might be similarly repressed in other polyploid organisms and at repetitive sequences in haploid and diploid species. Citation: Delmas S, Shunburne L, Ngo H-P, Allers T (2009) Mre11-Rad50 Promotes Rapid Repair of DNA Damage in the Polyploid Archaeon Haloferax volcanii by Restraining Homologous Recombination. PLoS Genet 5(7): e1000552. doi:10.1371/journal.pgen.1000552 Editor: Ivan Matic, Universite ´ Paris Descartes, INSERM U571, France Received October 10, 2008; Accepted June 9, 2009; Published July 10, 2009 Copyright: ß 2009 Delmas 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: We are grateful to the BBSRC, Wellcome Trust, MRC, and Fondation pour la Recherche Me ´dicale for funding and to the Royal Society for a University Research Fellowship awarded to TA. 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: thorsten.allers@nottingham.ac.uk ¤ Current address: Institute for Ageing and Health, Newcastle University, Newcastle Upon Tyne, United Kingdom Introduction Bacterial and eukaryotic cells are normally assumed to be haploid and diploid, respectively, but polyploidy is surprisingly widespread. Polyploid cells can arise naturally during development of otherwise haploid or diploid organisms (e.g. hepatocytes), or as a consequence of cellular stress and disease (e.g. cancer, reviewed in [1]). Organisms that are constitutively polyploid are common amongst eukaryotes, and include plants, fish and amphibians. Polyploid bacteria include the radiotolerant species Deinococcus radiodurans, which harbors ,8 copies of its genome [2], and Epulopiscium spp., which contain tens of thousands of genome copies [3]. Amongst archaea, Methanocaldococcus jannaschii, Halobac- terium salinarum and Haloferax volcanii have been shown to be naturally polyploid [4,5]. The presence of multiple genome copies affects many aspects of cell metabolism, in particular pathways of DNA repair. Since homologous recombination (HR) requires an identical genome copy, its usage for DNA repair is influenced by cell ploidy. When only one genome copy is present in the G1 phase of the eukaryotic cell cycle, DNA double-strand breaks (DSBs) are repaired by non- homologous end-joining (NHEJ), while HR is the predominant form of DSB repair in the G2 phase (reviewed in [6]). A further doubling of the ploidy of eukaryotic cells can result in increased reliance on HR, since genes involved in HR become essential for viability in tetraploid yeast [7]. In the presence of 8 genome copies in D. radiodurans, RecA-dependent HR is also required for DSB repair. However, HR is the second part of a two-stage DSB repair process, and is preceded by RecA-independent extended synthesis- dependent strand annealing [8]. It is a common assumption that additional genome copies might help protect polyploid cells from DNA damage. This is not the case, since tetraploid Saccharomyces cerevisiae cells are no more resistant to DNA damage than diploids [9,10]. Furthermore, D. radiodurans cells have the same survival rate after ionizing radiation, whether they contain 4 or 10 genome copies [11]. We have undertaken a study of DNA repair in the halophilic archaeon H. volcanii , which is naturally polyploid and contains 10–20 copies of the genome, depending on growth phase [4]. Archaea are of great interest in their own right, and share many core components of their DNA processing machinery with eukaryotes (reviewed in [12]). We have focused on the role of the Mre11-Rad50 complex, which is present in all domains of life and is involved in several pathways of DSB repair including HR and NHEJ (reviewed in [13]). Mre11 is a PLoS Genetics | www.plosgenetics.org 1 July 2009 | Volume 5 | Issue 7 | e1000552