DNA Helicase HIM-6/BLM Both Promotes MutSg-Dependent Crossovers and Antagonizes MutSg-Independent Interhomolog Associations During Caenorhabditis elegans Meiosis

Meiotic recombination is initiated by the programmed induction of double-strand DNA breaks (DSBs), lesions that pose a potential threat to the genome. A subset of the DSBs induced during meiotic prophase become designated to be repaired by a pathway that specifically yields interhomolog crossovers (COs), which mature into chiasmata that temporarily connect the homologs to ensure their proper segregation at meiosis I. The remaining DSBs must be repaired by other mechanisms to restore genomic integrity prior to the meiotic divisions. Here we show that HIM-6, the Caenorhabditis elegans ortholog of the RecQ family DNA helicase BLM, functions in both of these processes. We show that him-6 mutants are competent to load the MutSg complex at multiple potential CO sites, to generate intermediates that fulfill the requirements of monitoring mechanisms that enable meiotic progression, and to accomplish and robustly regulate CO designation. However, recombination events at a subset of CO-designated sites fail to mature into COs and chiasmata, indicating a pro-CO role for HIM-6/BLM that manifests itself late in the CO pathway. Moreover, we find that in addition to promoting COs, HIM-6 plays a role in eliminating and/or preventing the formation of persistent MutSg-independent associations between homologous chromosomes. We propose that HIM-6/BLM enforces biased outcomes of recombination events to ensure that both (a) CO-designated recombination intermediates are reliably resolved as COs and (b) other recombination intermediates reliably mature into noncrossovers in a timely manner. IN most eukaryotes, accurate segregation of homologous chromosomes during meiosis depends on crossover (CO) recombination events, as COs form the basis of connections known as chiasmata that help homologs orient toward opposite spindle poles at the meiosis I division (Page and Hawley 2003). Multiple mechanisms collaborate to guarantee that COs will form between every homolog pair. One level of regulation governs the initiation of recombination through the programmed formation of double-strand DNA breaks (DSBs), which form in substantial excess of eventual COs. Recent evidence suggests that checkpoint-like feedback mechanisms operate to ensure both that DSB formation continues until each homolog pair has at least one CO-eligible recombination intermediate and that DSB formation will shut down once this condition is met (Rosu et al. 2013; Stamper et al. 2013). Following DSB formation, a subset of the initial recombination intermediates is selected to become COs, recruiting a cohort of COpromoting (pro-CO) proteins that function to stabilize and protect these COdesignated intermediates (Baudat and De Massy 2007; Kohl Copyright © 2014 by the Genetics Society of America doi: 10.1534/genetics.114.161513 Manuscript received January 13, 2014; accepted for publication July 14, 2014; published Early Online July 21, 2014. Supporting information is available online at http://www.genetics.org/lookup/suppl/ doi:10.1534/genetics.114.161513/-/DC1. These authors contributed equally to this work. Present address: Department of Biology, Brooklyn College, The City University of New York, 2900 Bedford Ave., Brooklyn, NY 11210. Corresponding author: Department of Developmental Biology, 279 Campus Dr., B300 Beckman Center, Stanford University School of Medicine, Stanford, CA 94305. E-mail: [email protected] Genetics, Vol. 198, 193–207 September 2014 193 and Sekelsky 2013; Lynn et al. 2007). A widely conserved solution for protecting potential CO intermediates involves the MutSg complex, comprising MSH4 and MSH5, meiosisspecific members of the MutS protein family that can form a sliding clamp on DNA in response to recognition of branched DNA structures (Baudat and De Massy 2007; Lynn et al. 2007; Snowden et al. 2004). In many organisms, MutSg is initially recruited to multiple sites in excess of eventual COs, but it becomes stabilized at only a subset of these sites through recruitment of other pro-CO factors (Kneitz et al. 2000; Yokoo et al. 2012; Reynolds et al. 2013; Holloway et al. 2014; Qiao et al. 2014). The CO designation process is tightly regulated, yielding a highly nonrandom distribution in which a relatively small number of CO-based connections are formed between homologs, yet chromosome pairs lacking such connections are extremely rare. CO designation per se is not sufficient to ensure CO formation for every chromosome pair, however. Since the number of CO-designated sites in many organisms is on the order of one per chromosome (or one per chromosome arm), eventual resolution of the CO-designated intermediates must occur in a highly biased fashion, such that maturation of each CO-designated intermediate reliably yields a CO. Finally, excess recombination intermediates not destined for the CO fate must be faithfully repaired in a timely fashion to restore integrity of chromosomes prior to the meiotic divisions. In the current work, we investigate the roles of HIM-6, the Caenorhabditis elegans ortholog of the BLM DNA helicase (Wicky et al. 2004), in promoting successful meiosis. BLM is best known for its “anti-CO” activities and roles in antagonizing recombination in mitotically dividing cells. Mutations in the human Blm gene cause a familial cancer predisposition syndrome known as Bloom syndrome, and a diagnostic feature of Blm mutant patient cells is a highly elevated frequency of COs between sister chromatids (Chaganti et al. 1974; Ellis et al. 1995). Supporting the view of BLM as an anti-CO agent, BLM was identified as part of a protein complex that has an in vitro “dissolution” activity that can dismantle model recombination substrates containing double Holliday junctions in a manner that exclusively yields noncrossover products (Wu and Hickson 2003). Further, anti-CO roles during meiotic recombination have been demonstrated or proposed for BLM orthologs or its protein complex partners in a variety of species, including Saccaromyces cerevisiae, Arabidopsis, mice, and Drosophila (e.g., Rockmill et al. 2003; Jessop et al. 2006; Oh et al. 2007; Chelysheva et al. 2008; Holloway et al. 2010; Kohl et al. 2012). However, this reputation of BLM as an antagonist of crossing over was not readily reconciled with the finding that loss of function of him-6 results in a reduction of COs and chiasmata, implying a pro-CO rather than anti-CO role for BLM in C. elegans meiosis (Zetka and Rose 1995; Wicky et al. 2004). In the interim since HIM-6 was first identified as C. elegans BLM, substantial progress has been made in the C. elegans system both in identifying meiotic recombination machinery components acting at early and late steps and in development of in situmarkers for visualizing ongoing recombination events and other features of meiotic prophase progression. Here, we exploit these advances to revisit the roles of HIM-6/ BLM in meiotic recombination. We show that HIM-6/BLM has a role in promoting the formation of MutSg-dependent COs between homologous chromosomes that manifest itself late in meiotic prophase, and we show that HIM-6/BLM functions to ensure that CO-designated recombination intermediates reliably mature into interhomolog COs. Moreover, we show that in addition to its role in promoting meiotic CO formation, HIM-6/BLM plays a role in eliminating and/or preventing the formation of persistent MutSg-independent recombination-based interactions between the homologs. Our data suggest that HIM-6/BLM may function in multiple distinct contexts during meiosis, contributing both to ensuring the formation of COs and to restoring integrity of the chromosomes to enable their faithful segregation. This work complements and extends the findings of recent parallel studies investigating the requirements for resolution of meiotic CO intermediates in C. elegans (Agostinho et al. 2013; O’Neil et al. 2013; Saito et al. 2013) and contributes to a growing recognition that BLM can be deployed in a variety of different contexts to affect the structure of multiple classes of recombination intermediates and/or the timing and outcome of their resolution. The prominence of the pro-CO role of BLM during C. elegans meiosis suggests that the C. elegans system may be especially well suited for future studies addressing how the highly CO-biased outcome of resolution at CO-designated sites is accomplished. Materials and Methods