Mus81 nuclease and Sgs1 helicase are essential for meiotic recombination in a protist lacking a synaptonemal complex

9296–9309 Nucleic Acids Research, 2013, Vol. 41, No. 20 doi:10.1093/nar/gkt703 Published online 9 August 2013 Mus81 nuclease and Sgs1 helicase are essential for meiotic recombination in a protist lacking a synaptonemal complex Agnieszka Lukaszewicz, Rachel A. Howard-Till and Josef Loidl* Department of Chromosome Biology, Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, A-1030 Vienna, Austria Received May 7, 2013; Revised July 17, 2013; Accepted July 18, 2013 ABSTRACT Mus81 resolvase and Sgs1 helicase have wellestablished roles in mitotic DNA repair. Moreover, Mus81 is part of a minor crossover (CO) pathway in the meiosis of budding yeast, plants and vertebrates. The major pathway depends on meiosisspecific synaptonemal complex (SC) formation, ZMM proteins and the MutLc complex for COdirected resolution of joint molecule (JM)-recombination intermediates. Sgs1 has also been implicated in this pathway, although it may mainly promote the non-CO outcome of meiotic repair. We show in Tetrahymena, that homologous chromosomes fail to separate and JMs accumulate in the absence of Mus81 or Sgs1, whereas deletion of the MutLc-component Mlh1 does not affect meiotic divisions. Thus, our results are consistent with Mus81 being part of an essential, if not the predominant, CO pathway in Tetrahymena. Sgs1 may exert functions similar to those in other eukaryotes. However, we propose an additional role in supporting homologous CO formation by promoting homologous over intersister interactions. Tetrahymena shares the predominance of the Mus81 CO pathway with the fission yeast. We propose that in these two organisms, which independently lost the SC during evolution, the basal set of mitotic repair proteins is sufficient for executing meiotic recombination. INTRODUCTION Meiosis is the division by which germ progenitor cells reduce the somatic diploid chromosome set to the gametic haploid set. The chromosomes of the haploid set are mosaics assembled from corresponding parts of homologous parental chromosomes. The exchange of chromosome parts occurs by crossing over (CO). It contributes to the recombination of parental genes in the gametes and the genetic diversity of the offspring. At the same time, CO is instrumental in connecting homologous chromosomes by chiasmata, which are required for the correct bipolar orientation of bivalents during the first meiotic division. If CO is compromised, chromosomally unbalanced gametes may be formed. CO is induced by programmed DNA double-strand breaks (DSBs). At a CO site, one of the four chromatids of a chromosome pair experiences a DSB made by a dedicated endonuclease, Spo11 (1). The DSB is widened to a gap, and DNA flanking the DSB is resected in the 50 –30 direction, exposing single-stranded 30 overhangs. These single-stranded DNA ends associate with strand exchange proteins Rad51 and Dmc1, and one end invades a DNA double strand, which results in a threeway DNA structure, the so-called displacement loop (D-loop). If strands within the D-loop are complementary, they form a heteroduplex, and the invading strand extends by DNA synthesis (2). Most heteroduplexes seem to be short-lived and become unwound by helicases. Other D-loops capture the second DSB end and expand into a stable joint molecule (JM). The standard model of CO, elaborated in budding yeast, invokes a JM consisting of two Holliday junctions (HJs) (3). To disengage, JMs must be resolved by endonucleases. Depending on the cleavage orientation of the two HJs, the ligation of nicked strands may result in a reciprocal exchange of two DNA molecules, corresponding to a CO, or alternatively, in a nonreciprocal exchange, a noncrossover (NCO) (4,5). Based on their function in meiotic CO formation and their ability to cleave JMs in vitro, several potential eukaryotic HJ resolvases were identified. These were the Mlh1–Mlh3 (also known as MutLg) complex, the Rad1– Rad10 complex, the Mus81–Mms4/Eme1 complex, the Slx1–Slx4 complex and Yen1/GEN1 (6–8). Of these, the MutLg complex (with Mlh3 containing the nuclease motif) produces (in collaboration with Exo1) the majority of COs in budding yeast (9). The MutLg complex seems to work on JMs that are generated in the *To whom correspondence should be addressed. Tel: +43 1 4277 56210; Fax: +43 1 4277 9562; Email: ß The Author(s) 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Nucleic Acids Research, 2013, Vol. 41, No. 20 9297 context of a synaptonemal complex (SC) and the so-called ZMM proteins (Zip1,2,3, Msh4,5, Mer3, Spo16 and Spo22) (10,11). Paradoxically, Sgs1 (the BLM helicase ortholog), which has long been known for its mitotic anti-CO activity, functions as a CO promoting factor as well (9,12–14). There also exists a minor pathway in the budding yeast that is independent of ZMM proteins and MutLg, and involves the Mus81–Mms4 nuclease complex (and, to a lesser degree, Yen1). The fission yeast is believed to feature a nondouble HJ form of JM, either a single HJ (15) or a nonreciprocal (i.e. nicked HJ) CO precursor (16–18). JM resolution largely depends on the Mus81–Eme1 (the Mms4 ortholog) complex (16,19–21). Arabidopsis and mammals feature both a ZMM–MutLg-dependent pathway and a Mus81dependent pathway, with the former being predominant (22–24). Caenorhabditis elegans and Drosophila rely on different resolvase complexes (25,26). Because of the considerable diversity even within such a small selection of organisms, it is of interest to know whether other organisms use similar sets of resolvases or have come up with different solutions. Revealing the variability of CO pathways among different eukaryotes may help to understand the evolutionary flexibility of the meiotic process, and ultimately, the nature of primordial meiosis. To address these questions, we studied meiotic DSB processing in an evolutionarily distant model system, the ciliated protist Tetrahymena thermophila. Tetrahymena is a unicellular organism with two functionally distinct nuclei. One is the polyploid somatic macronucleus, which is transcriptionally active and is propagated only during the vegetative life cycle. The other is the transcriptionally silent micronucleus, which functions as the germ line. Only the micronucleus undergoes meiosis and is passed on to the offspring during sexual reproduction [(27) and Supplementary Figure S1]. Pairs of mating Tetrahymena cells undergo synchronous meioses (28), and the progression of meiosis can be easily followed and staged (Figure 1). Early steps in meiotic recombination follow the canonical pathway with Spo11 inducing DSBs, and strand exchange requiring Rad51 and Dmc1 (29,30). (...truncated)