Commentary 501 The choice in – defining the factors that influence crossover or non-crossover formation

Jillian L. Youds and Simon J. Boulton* DNA Damage Response Laboratory, Cancer Research UK, London Research Institute, Clare Hall, Blanche Lane, South Mimms EN6 3LD, UK *Author for correspondence ([email protected]) Journal of Cell Science 124, 501-513 © 2011. Published by The Company of Biologists Ltd doi:10.1242/jcs.074427

Summary Meiotic crossovers are essential for ensuring correct segregation as well as for creating new combinations of alleles for natural selection to take place. During meiosis, excess meiotic double-strand breaks (DSBs) are generated; a subset of these breaks are repaired to form crossovers, whereas the remainder are repaired as non-crossovers. What determines where meiotic DSBs are created and whether a crossover or non-crossover will be formed at any particular DSB remains largely unclear. Nevertheless, several recent papers have revealed important insights into the factors that control the decision between crossover and non-crossover formation in meiosis, including DNA elements that determine the positioning of meiotic DSBs, and the generation and processing of recombination intermediates. In this review, we focus on the factors that influence DSB positioning, the required for the formation of recombination intermediates and how the processing of these structures generates either a crossover or non-crossover in various organisms. A discussion of crossover interference, assurance and homeostasis, which influence crossing over on a chromosome-wide and -wide scale – in addition to current models for the generation of interference – is also included. This Commentary aims to highlight recent advances in our understanding of the factors that promote or prevent meiotic crossing over.

Key words: DSB repair, Meiosis, Crossover control,

Introduction overhangs that are initially bound by replication A (RPA), Meiosis is the specialised reductive division that generates haploid which is subsequently displaced by the recombinase radiation cells. During this process, a single round of replication is followed sensitive 51 (RAD51) and/or the meiosis-specific recombinase by two rounds of : in the first division dosage suppressor of Mck1 (DMC1) to form nucleoprotein (meiosis I), homologous segregate, whereas in the filaments. These filaments serve to find a complimentary sequence

Journal of Cell Science second division, sister chromatids segregate (meiosis II). A key within a homologous chromosome, at which they instigate single- step in meiosis I is the recognition of homologous chromosomes, end strand invasions (Hunter and Kleckner, 2001) to generate so- which then align and pair along the length of the chromosome. called displacement loop (D loop) recombination intermediates Once homologues have aligned, synapsis can proceed with the (Fig. 1). If the second end of the original DSB binds with the formation of the synaptonemal complex (SC), a protein structure homologous chromosome, a double is formed, that supports and maintains homologues in close juxtaposition and which can be resolved to generate either a non-crossover or an serves as a scaffold for crossover-promoting recombination factors. interhomologue crossover, the latter of which is hereafter referred Meiotic crossing over involves the generation of meiotic double- to simply as crossover (Bishop and Zickler, 2004; Schwacha and strand breaks (DSBs), which are subsequently repaired either as Kleckner, 1995). Double Holliday junctions can also be processed crossovers or non-crossovers (Fig. 1). Meiotic recombination is through dissolution, which results in a non-crossover (Fig. 1) (Wu not only necessary to create new allele combinations that generate and Hickson, 2003). Meiotic non-crossovers have also been genetic diversity, but is also essential in ensuring accurate proposed to form when strand invasion is transient, and when a chromosome segregation at the first meiotic division because the limited amount of DNA synthesis occurs before the invaded strand crossover acts as a tether between homologues, which ensures that dissociates and anneals to its partner strand, as in mitotic synthesis- each homologue will properly align at the metaphase plate and dependent strand annealing (SDSA; see Box 1 and Fig. 1) (Allers thereby correctly attach to the spindle. DSB repair occurs and Lichten, 2001; Bishop and Zickler, 2004). During meiosis in concurrently with SC formation and is required for normal synapsis budding yeast, non-crossover heteroduplex products are found to in yeast and mice (Baudat et al., 2000; Roeder, 1997; Romanienko form with the same timing as double Holliday junctions, whereas and Camerini-Otero, 2000), whereas in crossovers occur later (Allers and Lichten, 2001), consistent with and Drosophila melanogaster, homologue pairing and SC formation the idea that crossovers and non-crossovers are formed through can occur independently of meiotic recombination (Colaiacovo et distinct pathways. Analysis of ZMM mutants (Zip1, Zip2, Zip3, al., 2003; Dernburg et al., 1998; Liu et al., 2002; McKim et al., Zip4, Mer3, Msh4 and Msh5; further discussed below) in yeast 1998). indicates that the decision between crossover and non-crossover is The process of meiotic recombination is initiated when meiotic made very early, i.e. at or prior to the establishment of a stable DSBs are created by the endonuclease SPO11, in conjunction with single-end invasion intermediate, when one of the two ends of the a number of additional proteins (Keeney and Neale, 2006). DSBs DSB invades its homologous chromatid (Bishop and Zickler, 2004; are then resected to generate 3Ј single-strand DNA (ssDNA) Borner et al., 2004; Hunter and Kleckner, 2001). Thus, the 502 Journal of Cell Science 124 (4)

Double-strand-break formation

Single-end invasion

3Ј 3Ј 5Ј 5Ј 5Ј 3Ј

D loop recombination intermediate

Double Holliday junction Fig. 1. Model for meiotic crossover or non-crossover formation. Double strand breaks are generated and their 5Ј ends are resected to generate a 3Ј overhang. A strand invasion event then generates a single-end invasion D loop intermediate. If the second end of the original DSB also engages with the homologue, a double Holliday junction is formed (shown on the left). The double Double Holliday Double Holliday SDSA Holliday junction can be resolved to form either a junction resolution junction dissolution crossover (interference-dependent) or a non-crossover. Alternatively, the junction can be dissolved by double Holliday junction dissolution to form a non-crossover. Instead of forming a double Holliday junction, the D Crossover Non-crossover Crossover loop can be dissociated and the invading strand can (interference-dependent) (interference-independent) or associate with the opposite end of the original break, as in synthesis-dependent strand annealing (SDSA), to form a non-crossover. Alternatively, the intermediate can be acted upon by enzymes such as Mus81 that can form Non-crossover Non-crossover interference-independent crossovers.

Journal of Cell Science crossover–non-crossover decision is thought to occur around the Several studies have shown that Spo11 access to DNA is one of time of strand exchange. the determinants of DSB location, because DSBs are often found This Commentary will discuss the factors that contribute to within open chromatin in yeast (Berchowitz et al., 2009; Ohta et crossover or non-crossover formation in meiosis, including the al., 1994; Wu and Lichten, 1994). In C. elegans, the putative generation and positioning of meiotic DSBs, formation of the SC chromatin-modifying protein high incidence of males 17 (HIM- and generation of recombination intermediates. We will also discuss 17) is required for successful DSB formation and for normal levels how interhomologue crossing over is promoted compared with of dimethylation of lysine residue 9 of histone 3 (H3K9Me2) intersister repair, how recombination intermediates are resolved during meiosis, because mutants display a marked reduction in into crossovers as well as how anti-recombinases prevent crossing H3K9Me2 staining (Reddy and Villeneuve, 2004). Furthermore, over. Crossover interference, assurance and homeostasis will also DSB formation in him-17 non-null mutants can be enhanced by be discussed (see text box), including a summary of the current loss of LIN-35, the C. elegans retinoblastoma (Rb) protein models for how crossover interference is established. homologue, which is a known component of chromatin modifying complexes (Reddy and Villeneuve, 2004). Collectively, these data Control of meiotic DSB formation suggest that the activity of HIM-17 alters the chromatin state, Meiotic crossovers tend to form at specific sites in the genome of probably by opening the chromatin to allow access by SPO-11, most organisms (Buard and de Massy, 2007; Mezard, 2006; Pryce and this governs DSB competence. More recently, components of and McFarlane, 2009); these are known as recombination hotspots. the C. elegans condensin I complex have been implicated in In mammals, hotspots are typically regions of 1–2 kb that occur regulating both the position and number of DSBs, because mutants every 50–100 kb within the genome (Jeffreys et al., 2001; McVean, show increased formation of meiotic DSBs and a distribution of 2010; Myers et al., 2008). Human recombination hotspots often crossovers that differs from the wild type (Mets and Meyer, 2009; occur relatively close to (within 50 kb), but are preferentially Tsai et al., 2008). Here, it was demonstrated that disruption of the found outside of transcribed regions (Myers et al., 2005). At condensin I complex causes an increase in chromosomal axis hotspots in eukaryotes, meiotic DSBs are generated by the length, which is independent of DSB formation but requires the conserved topoisomerase-like endonuclease Spo11 (Bergerat et al., axis-associated protein HIM-3 (Mets and Meyer, 2009). Thus, in 1997; Cao et al., 1990; Keeney et al., 1997). However, insights C. elegans, it appears that the condensin I complex controls into what controls the position of break formation were unknown chromosome structure and the association of chromatin with the until recently. chromosome axis, which in turn might determine its accessibility Meiotic crossover and non-crossover control 503

by SPO-11. This and other evidence suggests that the chromosome axis has an important role in meiotic DSB formation. Certainly, Box 1. Terms used to describe meiosis proteins associated with the chromosome axis have also been Anti-recombinase: A protein that acts to prevent or inhibit shown to be important for DSB formation. For example, the C. recombination. Chiasmata: The (cytologically) visible structure that is the elegans HIM-3 paralog – him three paralog 3 (HTP-3) – is a physical manifestation of a crossover between homologous component of the meiotic chromosome axis that mediates DSB chromosomes in meiosis. formation through its interaction with a complex required to Crossover assurance: The mechanism that makes certain each generate DSBs (Goodyer et al., 2008). The conserved mouse homologous chromosome pair will receive at least one meiotic protein meiosis-specific 4 (MEI4), which associates with the axis crossover. of meiotic chromosomes, was also shown to be required for DSB Crossover homeostasis: A mechanism that ensures a constant formation (Kumar et al., 2010). Thus, a functional relationship number of meiotic crossovers, even under conditions where more appears to exist between the chromosome axis and DSB formation or fewer meiotic DSBs are generated. Homeostasis maintains and might have some bearing on where hotspots arise. For a meiotic crossovers at the expense of non-crossovers. How detailed discussion and models of the interplay between the homeostasis is maintained is not well understood but it might be governed by the same mechanism as interference. chromosome axis and DSB formation, readers are referred to the Crossover interference: A mechanism that distributes meiotic article by Kleckner (Kleckner, 2006). DSBs and crossovers such that adjacent crossovers tend to If DSB hotspots are associated with sites of open chromatin, occur further apart than expected by chance. The basis of then the question arises what exactly determines the chromatin crossover interference is not well understood. state at hotspots. Evidence suggests that some recombination D loop: A recombination intermediate structure wherein one end hotspots in humans are associated with specific sequence motifs. of the DSB has invaded into the homologous chromosome and Ten percent of the hotspots identified in a genome-wide survey of been extended to form a stable structure. A D loop can be ~1.6 million single-nucleotide polymorphisms (SNPs) in three dissociated to allow non-crossover repair through SDSA, or can sample populations were associated with the 7-mer motif go on to form a double Holliday junction. See Fig. 1. CCTCCCT (Myers et al., 2005). Using more detailed data from Double Holliday junction: A recombination intermediate structure that can be formed following D loop formation if the the human ‘haplotype map’ (HapMap) project, work by the second end of the original DSB also associates with the same group identified a degenerate 13-mer sequence homologous chromosome. This structure can be processed into (CCNCCNTNNCCNC) that was present in one or more copies at either a crossover or non-crossover. See Fig. 1. 40% of all human hotspots (Myers et al., 2008). These data indicate Haplotype map: A map generated to describe common patterns that there is some correlation between DNA sequence and hotspot of human genetic variation using single nucleotide location. However, although humans and chimpanzees have more polymorphisms in various populations. Also known as the than 98% sequence identity, hotspot locations in these two species HapMap. are not conserved (Ptak et al., 2005; Winckler et al., 2005). In Interference-independent crossovers: Crossovers that do not addition, there is evidence that recombination rates vary both exhibit interference. Crossovers in this class are formed by between ethnic groups (Evans and Cardon, 2005) and between Mus81 catalyzed recombination intermediate cleavage in many organisms. Because they do not display interference, Journal of Cell Science individuals (Cheung et al., 2007; Coop et al., 2008). Thus, sequence interference-independent crossovers can occur in close proximity. alone is clearly not the only factor determining hotspot activity. Interference-independent crossovers contrast with interference- Recent studies indicate a role for specific epigenetic dependent crossovers, which always display interference in terms modifications at DSB sites. A recent study in C. elegans identified of their positioning. the new chromatin factor X-non-disjunction factor 1 (XND-1), Interhomolog crossover: Also known as a meiotic crossover, a which is required for DSB formation specifically on the X crossover that has occurred between homologous chromosomes chromosome, as well as for the distribution of DSB formation (but during meiosis. not DSB number) on the autosomes (Wagner et al., 2010). Increased Intersister repair: Repair of damage or a meiotic DSB (as in this acetylation of histone H2A at lysine 5 (H2AK5Ac) that is likely to article) that uses the sister chromatid as a template rather than be mediated by the histone acetylation protein Myst family histone the homologous chromosome. Repair between sister chromatids can be through crossover or non-crossover pathways. acetyltransferase-like (MYS-1; TIP60 in humans) in xnd-1 mutants Synthesis-dependent strand annealing (SDSA): A pathway for was identified as a chromatin modification associated with the non-crossover repair wherein a D loop recombination alteration in autosomal distribution of meiotic DSBs and reduced intermediate is formed and limited DNA synthesis occurs in the DSB formation on the X chromosome (Wagner et al., 2010). This region of the break, using the homologous chromosome as a study suggests that H2AK5Ac is a determinant of DSB distribution template. The D loop is then dissociated and the invading end of in C. elegans, although the influence of this modification on DSB the DSB that has been extended anneals back with the other end formation in other organisms still needs to be shown. Trimethylation of the original DSB. DNA synthesis and ligation seals the break. of lysine 4 of histone 3 (H3K4Me3) has been considered as a pre- ZMM proteins: A group of proteins that includes Zip1, Zip2, Zip3, existing marker for sites of DSB formation in S. cerevisiae because Zip4, Mer3, Msh4, Msh5 and the recently identified Spo16 in S. this histone modification is frequently found in regions close to cerevisiae; they are involved in both SC formation and meiotic crossing over. DSB sites, independently of expression levels (Borde et al., 2009). The same study showed that deletion mutants of the SET- domain-containing 1 (set1) gene, which encodes the only H3K4 initiation sites in mice with H3K4Me3 enriched at active DSB methyltransferase in S. cerevisiae, display a dramatic reduction in sites, whereas histone H4 hyperacetylation was found to occur DSBs, and that those DSBs that form in the absence of Set1 are after DSB formation (Buard et al., 2009). H3K4Me3 is present in differentially localised (Borde et al., 2009). Specific histone Spo11–/– mice, which lack meiotic DSBs, and thus H3K4Me3 is a modifications were also shown to be present at meiotic DSB marker for sites of DSB initiation that might contribute to hotspot 504 Journal of Cell Science 124 (4)

activity (Buard et al., 2009). HapMap-methylation-associated SNPs, likely to account for all hotspot activity. As the roles of epigenetic which are markers of germline methylation, are positively correlated modifications in meiotic DSB formation and crossover control with regional meiotic recombination rates in humans (Sigurdsson remain poorly understood, they should be an important focus of et al., 2009), providing further evidence that epigenetic future investigation. modifications influence hotspot activity in mammals. It remains unclear exactly how sites of frequent meiotic Proteins that influence crossover outcomes recombination are identified and marked as hotspots. However, The factors influencing DSB position are not yet well-understood, recent studies in mice have identified the gene PR-domain- but a number of proteins that function downstream of DSB creation, containing 9 (Prdm9) (Baudat et al., 2010; Parvanov et al., 2010), to either promote or prevent crossover formation, have been which encodes for a protein with histone methyltransferase activity. characterised. Although it is not known exactly how the decision Different human alleles of PRDM9 show altered activity of is made to form a crossover or non-crossover, many meiotic recombination hotspots (Baudat et al., 2010). Furthermore, in vitro, proteins influence whether or not a crossover can take place. Here, the protein product of the human PRDM9 A allele was shown to these proteins will be classified as having either pro-crossover or bind to the specific 13-mer motif previously associated with anti-crossover activities, although at least one of these proteins can recombination hotspots, suggesting that PRDM9 marks DSB be considered to fit into both categories, depending on the context initiation sites by recognising this motif (Baudat et al., 2010). of the protein activity or the model system in which it has been Thus, sequence motifs that are modified by certain epigenetic examined. A non-exhaustive list of these genes is presented in marks appear to designate some of the known mammalian Table 1. recombination hotspots by either signalling to the recombination machinery or allowing SPO11 to access the DNA. However, it is Pro-crossover proteins not known how these signals integrate with factors that control the Setting the stage for crossover formation association of chromatin with the chromosome axis. Furthermore, A number of pro-crossover proteins have roles in pairing or SC the H3K4Me3 mark is highly enriched in promoter regions of formation that indirectly promote crossovers. SC formation involves actively transcribed genes (Bernstein et al., 2005; Schneider et al., the assembly of several structural elements, specifically a pair of 2004), but meiotic DSBs do not form in all promoter regions and, lateral elements connected by transverse elements and a central currently, there is no known direct relationship between DSB element, which facilitates crossovers by continuously maintaining formation and transcriptional activity (Hunter, 2006; Kniewel and homologues in close juxtaposition (Costa and Cooke, 2007; de Keeney, 2009). Thus, this chromatin modification alone is not Carvalho and Colaiacovo, 2006). Among the proteins that form the

Table 1. A non-exhaustive list of genes with either pro- or anti-crossover activities S. pombe S. cerevisiae C. elegans Drosophila Arabidopsis Vertebrates Pro-crossover activity DSB end resection rad32, rad50, mre11, rad50, mre-11, rad-50 mre11, rad50, Atmre11, MRE11, RAD50, NBS1 nbs1 (complex) xrs2 (complex) nbs Atrad50, Atnbs1 (complex)a

Journal of Cell Science ctIP sae2 com-1 CG5872?a Atgr1/Atcom1 CtIPa rqh1 sgs1 him-6 dmBlm Atrecq4a BLMa exo1 exo1 exo-1 tos ? EXO1a dna2 dna2 -2 ?? DNA2a Crossover intermediate rhp51 rad51 rad-51 spnB Atrad51 RAD51a formation dmc1 dmc1 absent absent Atdmc1 DMC1 rad22 rad52 brc-2 brca2 Atbrca2 BRCA2a mer3 Atmer3/rck rhp54 rad54 rad-54 rad54 Atrad54 RAD54a absent , msh5 msh-4/him-14, absent Atmsh4, Atmsh5 MSH4, MSH5 msh5 mlh1 mlh1 mlh-1a mlh1 Atmlh1 MLH1 mlh3 Atmlh3 MLH3 Promote interhomolog hop1 hop1 crossing over rad3a mec1 atl-1a mei-41 Atatra ATRa rad3a tel1 atm-1a atm/tefua Atatma ATM a mek1 mek1 rad50a rad50a rad-50 rad50a Atrad50a RAD50a Crossover intermediate mus81, mus81, mms4 mus-81, eme-1 mus81, mms4 Atmus81 MUS81, EME1 processing (complex) (complex) (complex) (complex) (complex) rad16b rad1b him-9 mei-9 Atrad1b XPF b absent yen1 gen-1b gen1a ? GEN1a slx1, slx4 slx1, slx4 slx-1, him-18 slx-1, mus312 ? SLX1, SLX4/BTBD12a (complex) (complex) (complex) (complex) (complex) Anti-crossover activity dHJ dissolution rqh1 sgs1 him-6a dmBlma Atrecq4aa BLM a D-loop dissociation absent absent rtel-1 CG4078?a ? RTEL1a

aHomologs that exist but have not yet been characterized with respect to the meiotic activity indicated. bCharacterized homologs that do not appear to have a role in meiosis. Atgr1, Arabidopsis thaliana -response gene 1; tos, Drosophila tosca; spnB, Drosophila spindle-B; rck, Arabidopsis rock-n-rollers; mlh3, Mut L homolog 3; tefu, Drosophila telomere fusion; mms4, methyl methanesulfonate sensitive. Meiotic crossover and non-crossover control 505

SC in S. cerevisiae are the Zip proteins (part of the ZMM group of distinct sites (Sharan et al., 1997; Thorslund et al., 2007), and is proteins). Zip1 is a structural protein that makes up the central required for localisation of RAD51 and DMC1 at foci that are regions of the SC (Sym et al., 1993). Zip2, Zip3 and Zip4 are also presumed to be meiotic DSBs in mice (Sharan et al., 2004). Similarly, required for SC assembly, and have been implicated in C. elegans BRCA2 (BRC-2) is essential for meiotic DSB repair, in ubiquitylation and/or sumoylation of proteins associated with SC which it functions to promote RAD-51 filament nucleation and formation (Agarwal and Roeder, 2000; Borner et al., 2004; Cheng stabilisation, and in the stimulation of RAD-51 mediated strand et al., 2006; Chua and Roeder, 1998; Perry et al., 2005; Tsubouchi exchange (Martin et al., 2005; Petalcorin et al., 2007; Petalcorin et et al., 2006). A common theme among SC proteins is that, although al., 2006). the sequence homologues of lower-organism SC proteins are not Mismatch repair defective 4 and 5 (Msh4 and Msh5, respectively) easily detectible in higher organisms, structural features of the and meiotic recombination 3 (Mer3), i.e. other members of the proteins are conserved (Costa and Cooke, 2007). In mammals, the ZMMs, also promote crossovers, probably by facilitating the stable SC is made up of multiple proteins including SC proteins 1, 2 and formation of recombination intermediates. msh4 and msh5 mutants 3 (SYCP1, 2 and 3), SC central element proteins 1 and 2 (SYCE1 in S. cerevisiae have reduced inter-homologue crossing over and SYCE2) and testis-expressed gene 12 (TEX12) (Costa and (Hollingsworth et al., 1995; Ross-Macdonald and Roeder, 1994). Cooke, 2007), but these will not be discussed further here. A Similarly, in C. elegans, MSH-4 and MSH-5 are essential for detailed review of proteins that are involved in homologue pairing crossing over (Kelly et al., 2000). Msh4 and Msh5 are known to and synapsis, as well as recent insights into meiotic pairing centres form a heterodimer, and one proposal is that this acts as a clamp to in C. elegans that are beyond the scope of this review can be found hold homologous chromosomes together, thereby stabilising the elsewhere (Costa and Cooke, 2007; Ding et al., 2010; Lynn et al., Holliday junction and facilitating crossing over (Snowden et al., 2007; Yang and Wang, 2009; Zetka, 2009; Zickler, 2006). 2004) (Fig. 2). In Msh4 and Msh5 knockout mice, chromosomes fail to correctly pair, crossovers are absent and, consequently, Generation of the recombination intermediate animals are sterile (de Vries et al., 1999; Edelmann et al., 1999; In the last few years, considerable progress has been made towards Kneitz et al., 2000). In mammals, unlike in S. cerevisiae, Msh4 and defining the activities that process DSBs to generate 3Ј ssDNA Msh5 are required for correct chromosome pairing during meiotic overhangs, which are the substrate for initiating homologous prophase, and both are clearly associated with recombination recombination. Studies of C. elegans completion of meiotic intermediates destined to form both crossovers and noncrossovers recombination-1 (com-1) and its homologue in Arabidopsis thaliana, (Kneitz et al., 2000; Santucci-Darmanin et al., 2000). Thus, the com1 (homologues of yeast sae2, human CtIP), found a similar mouse phenotype associated with loss of Msh4 or Msh5 does not phenotype in these two species, in which mutants failed to load only reflect of a loss of crossovers, but is also more severe than that RAD51 onto meiotic DSBs, suggesting that COM1 acts in DSB of other mutants, such as loss of the mut L homologue 1 (Mlh1; resection (Penkner et al., 2007; Uanschou et al., 2007). More recently, discussed below) (Edelmann et al., 1996). Interestingly, msh4 and data from budding yeast and mammalian cells have revealed that msh5 are not found in D. melanogaster and in S. pombe. The latter DSB resection is dependent on the cooperative action of multiple relies solely on the structure-specific endonuclease complex between factors, including the Mre11–Rad50–Xrs2 (MRX) complex [Mre11– mutagenesis sensitive 81 (Mus81) and essential meiotic

Journal of Cell Science Rad50–Nbs1 (MRN) in mammals] SUMO activating enzyme 2 endonuclease 1 (Eme1) for crossover formation (Osman et al., (Sae2; CtIP in mammals), small growth suppressor 1 [Sgs1; Bloom’s 2003), whereas in D. melanogaster the clamp function of Msh4 and syndrome mutated (BLM) in mammals], exonuclease 1 (Exo1; Msh5 can potentially be replaced by alternative proteins or an EXO1 in mammals) and DNA replication helicase 2 (Dna2; DNA2 alternative mechanism might be present (Blanton et al., 2005). In in mammals) (Gravel et al., 2008; Mimitou and Symington, 2008; addition to Msh4 and Msh5, the Mer3 helicase promotes normal Zhu et al., 2008) (Fig. 2). These studies have been validated by the crossover frequencies in yeast (Nakagawa and Ogawa, 1999). Mer3 analysis of DSB end processing in vitro using the respective purified functions to stimulate heteroduplex extension by Rad51, thereby proteins (Nimonkar et al., 2008). This area has been intensively stabilising nascent D loop structures to promote capture of the reviewed and discussed elsewhere and we refer the reader to several second free DNA end, double Holliday junction formation and, reviews on this topic (Bernstein and Rothstein, 2009; Mimitou and ultimately, crossovers (Mazina et al., 2004). In Sordaria, Mer3, Symington, 2009). Once the DSB has been processed, Rad51 and/or Msh4 and Mlh1 have recently been shown to have roles in the meiosis-specific recombinase Dmc1 bind the 3Ј ssDNA to homologous chromosome pairing (Storlazzi et al., 2010). generate a nucleoprotein filament that carries out a homology search MLH1 is a well-known marker for sites designated as crossovers and strand invasion into the homologous chromosome. Both Dmc1 in the mouse (Anderson et al., 1999). MLH1 is required for and Rad51 are required for efficient meiotic recombination in yeast crossovers, as germ cells from mice that lack MLH1 do not display (Schwacha and Kleckner, 1997) and mice (Pittman et al., 1998; sufficient chiasmata and do not progress beyond the meiotic Sharan et al., 2004; Yoshida et al., 1998), but Dmc1 orthologues pachytene stage, and thus, Mlh1 null mice are infertile (Edelmann have not been identified in C. elegans or D. melanogaster. Human et al., 1996) (Fig. 3). Budding yeast mlh1 deletion mutants have DMC1 has robust D-loop-forming activity in vitro (Li et al., 1997; reduced crossing over (Hunter and Borts, 1997). In vitro, human Sehorn et al., 2004), whereas in S. cerevisiae, Rad54 is required MSH4 interacts with MLH1, and in vivo the two proteins colocalise together with Rad51 for D loop formation (Sung et al., 2003). In during early to mid-pachytene, when crossing over takes place vivo, the Dmc1 nucleoprotein filament is more adept at forming D (Santucci-Darmanin et al., 2000). One possible function of MLH1 loops with the homologous chromosome than the Rad51 is that it mediates release of the MSH4 and MSH5 clamp (Snowden nucleoprotein filament (Shinohara et al., 2003; Tsubouchi and Roeder, et al., 2004), thereby facilitating crossover completion. mlh-1 has 2003). Co-ordination of RAD51 and DMC1 activities in mammalian not been characterised in C. elegans; instead, Zip homologous meiosis might be achieved through the breast cancer susceptibility protein 3 (ZHP-3), the C. elegans homologue of yeast Zip3, has protein 2 (BRCA2), because BRCA2 binds to both proteins at been shown to be a marker for meiotic crossovers (Fig. 3). ZHP-3 506 Journal of Cell Science 124 (4)

Enzyme Activity Outcome

RTEL-1 Non-crossover Mph1 Strand ? displacement

Sgs1 Pro-recombination Rqh1 DSB resection Helicases HIM-6 MUS309 BLM Non-crossover Dissolution

MER3 Pro-crossover Heteroduplex extension

Mus81 Crossover Strand MEI9 (XPF) cleavage Crossover Yen1/GEN1 Crossover and/or SLX4, HIM-18, Resolution non-crossover MUS312, BTBD12

DNA- MLH1/3 ? binding Pro-crossover proteins MSH4/5

Journal of Cell Science Fig. 2. Biochemical activities that promote crossover and/or non-crossover recombination. Shown are schematic representations of specific recombination intermediates that are subject to biochemical activities of meiotic enzymes (helicases, nucleases, DNA-binding proteins) that act to promote one of two repair outcomes: non-crossover or crossover (recombination). Arrows indicate the directionality of the helicase activity; arrows with scissors indicate the position of cleavage of the activity; yellow circles indicate the preferred substrate for DNA binding. The human BLM orthologues Sgs1 (S. cerevisiae), Rqh1 (S. pombe), HIM-6 (C. elegans) and Mus309 (D. melanogaster) are presumed to perform roles in both resection of DSBs and dissolution of double Holliday junctions. Yen1 is the S. cerevisiae orthologue of human GEN1. Slx4, HIM-18 and Mus312 are the S. cerevisiae, C. elegans and D. melanogaster orthologues of human BTBD12, respectively.

has two roles in meiosis: it promotes crossover formation and also homologues but not between sister chromatids. For example, in restructures chromosomes at diakinesis, the chromosome budding yeast, interhomologue recombination is ensured by condensation stage, so that appropriate segregation can take place phosphorylation of the axial element protein homologue pairing 1 (Bhalla et al., 2008; Jantsch et al., 2004). Thus, several proteins are (Hop1) by the serine/theronine protein kinases meiotic checkpoint required to generate and maintain a stable recombination 1 (Mec1) and telomere length 1 (Tel1), the homologues of intermediate. In order for appropriate chromosome segregation to mammalian ataxia telangiectasia and Rad3-related protein (ATR) take place, the stable recombination intermediate must have and ataxia telangiectasia mutated (ATM), respectively (Carballo et engaged the homologous duplex and not the sister chromatid, al., 2008). It has been proposed that Hop1 phosphorylation leads ultimately resulting in formation of an interhomologue crossover. to dimerisation of the meiotic axial element meiotic kinase 1 (Mek1), which enables it to phosphorylate its target proteins that Promoting interhomologue crossing over versus intersister prevent the repair of DSBs using the sister chromatid (Niu et al., repair 2005). In addition, Mek1 inhibits Rad51 activity by attenuating the Meiotic crossover formation can be controlled by genes that allow formation of the Rad51–Rad54 complex; with less active Rad51, meiotic DSB repair from only certain templates, i.e. not all. The the activity of the meiosis-specific strand exchange protein Dmc1 proteins that participate in the barrier to sister chromatid repair are is favoured (Niu et al., 2009). Dmc1 is thought to be more efficient pro-crossover factors because they promote DSB repair that uses at promoting interhomologue recombination than Rad51 (Shinohara the homologous chromosome, but not the sister chromatid (see text et al., 2003; Tsubouchi and Roeder, 2003), thus, the activity of box). In this way, these factors allow crossovers between Mek1 promotes interhomologue recombination over sister Meiotic crossover and non-crossover control 507

A Mouse crossover sites marked by MLH1 Resolution of the recombination intermediate Crossover formation is controlled directly by enzymes that act on recombination intermediates and resolve these either as crossovers or non-crossovers. Mus81–Eme1 can resolve intermediates into crossovers (Fig. 2), but also has multiple biochemical abilities, including a preference for acting on structures that include D loops, nicked Holliday junctions, replication forks with the lagging strand at the junction point, and 3Ј flap structures (Bastin-Shanower et al., 2003; Fricke et al., 2005; Osman et al., 2003). Yeast Mus81– Eme1 is also reported to have robust cleavage activity on intact Holliday junctions, although they are not its preferred substrate in vitro (Gaskell et al., 2007). Mus81 is responsible for generating interference-independent crossovers in S. cerevisiae (Argueso et SCP3 MLH1 al., 2004; de los Santos et al., 2003) and in mouse (Holloway et al., 2008), and also generates the majority of crossovers in S. B C. elegans crossover sites marked by ZHP-3 pombe, which has only interference-independent crossovers (Boddy et al., 2001; Osman et al., 2003; Smith et al., 2003). In C. elegans, DAPI + GFP GFP only MUS-81 is also responsible for a subset of crossovers that occur when meiotic non-crossover repair through SDSA is blocked by mutation in the anti-recombinase regulator of telomere length 1 Wild type (RTEL-1) (Youds et al., 2010). A number of DNA processing enzymes have recently been identified in various organisms that possess the biochemical activity to resolve Holliday junctions – specifically, the ability to cleave Holliday junctions symmetrically. These resolvases have the potential to generate either crossover or non-crossover products, rtel-1 mutant and other factors, perhaps the MSH4-MSH5 complex, might influence this decision. The human resolvase XPG-like endonuclease 1 (GEN1), and its S. cerevisiae orthologue Yen1, have been independently identified through their ability to Fig. 3. Examples of crossover interference. (A)Examples of crossovers symmetrically cleave Holliday junctions (Ip et al., 2008) (Fig. 2). marked by staining of MLH1 (green) on mouse chromosomes. The SC is Yen1 has functional overlap with Mus81 in budding yeast (Blanco marked in red by staining of the SC protein 3 (SCP3). When multiple et al., 2010) and human GEN1 was found to rescue the meiotic crossovers occur on the same chromosome, they tend to be spaced far apart, phenotype of mus81 S. pombe mutants (Lorenz et al., 2009), demonstrating crossover interference. (B)Complete crossover interference in Journal of Cell Science indicating conserved, functionally similar roles for Mus81 and C. elegans. The images in the top row show the single recombination foci per Yen1 or GEN1 in yeast and humans, respectively. However, we chromosome marked by staining of ZHP-3 (six chromosomes, hence six foci) at meiotic diplotene in a wild type animal. The rtel-1 mutant exhibits defective have not observed a meiotic phenotype for gen-1 mutants in C. complete crossover interference, as illustrated by the increased number of elegans (J.L.Y. and S.J.B., unpublished data) and, together with the ZHP-3 foci per meiotic nucleus (bottom images). recently demonstrated role for gen-1 in DNA damage signalling and repair in the nematode (Bailly et al., 2010), this indicates that additional enzyme(s) act as the meiotic resolvase in this organism. chromatid repair. In C. elegans – which do not have Dmc1 – germ In addition to GEN1, concurrent works from several groups cells, at the onset of meiotic prophase, switch into a specialised described roles for the orthologous proteins Drosophila Mus312, mode of DSB repair that is characterised by the requirement for C. elegans HIM-18, and mammalian synthetic lethal of unknown RAD-50 in loading RAD-51 onto meiotic DSB ends, a process function 4 (SLX4) in resolving Holliday junctions (Andersen et that is essential for interhomologue crossover formation (Hayashi al., 2009; Fekairi et al., 2009; Munoz et al., 2009; Saito et al., et al., 2007). By mid- to late pachytene, a second developmentally 2009; Svendsen et al., 2009) (Fig. 2). In mus312 mutants of D. programmed switch occurs; RAD-50 is no longer required for melanogaster, meiotic crossing over is reduced by ~95%, consistent RAD-51 association with DSBs, and competence for with the hypothesis that Mus312 is the key protein required for interhomologue crossing over is lost (Hayashi et al., 2007). Repair Holliday junction resolution in this organism (Andersen et al., of any remaining DSBs might then occur through the sister 2009; Yildiz et al., 2002). Gene expression patterns and knockdown chromatid, which requires BRCA homologue 1 (BRC-1) that is studies suggest that mammalian SLX4 has a meiotic function in dispensable for repair through the homologue (Adamo et al., 2008). humans that is conserved with that of D. melanogaster Mus312 The requirement for RAD-50 in RAD-51 loading at DSBs is (Andersen et al., 2009). SLX4 interacts with SLX1, and the SLX4– partially dependent on a number of proteins that have roles in SLX1 complex has a robust Holliday junction cleavage activity in meiosis-specific chromosome axis structure (Hayashi et al., 2007). vitro, which probably accounts for its role in meiosis (Fekairi et Thus, mechanisms exist to ensure that crossing over with the al., 2009; Munoz et al., 2009; Svendsen et al., 2009). SLX4 also homologue will occur at the correct time, rather than with the sister binds the structure-specific complex between the endonucleases chromatid. Once the stable interhomologue crossover intermediate Xeroderma Pigmentosum complementation group F and excision has been generated, the activity of a resolution enzyme will repair cross-complementing 1 (XPF–ERCC1) complex and ultimately complete the crossover. MUS81–EME1, among other proteins. Depletion of SLX4 also 508 Journal of Cell Science 124 (4)

causes sensitivity to a number of DNA damage agents, indicating RTEL1 are the only examples of meiotic anti-crossover proteins in that it serves as a platform for different structure-specific the current literature. However, it has been reported that S. endonucleases that are most likely to act both in meiosis and in cerevisiae mutator phenotype 1 (Mph1) has biochemical abilities DNA repair (Fekairi et al., 2009; Munoz et al., 2009; Svendsen et similar to those of RTEL1 (Prakash et al., 2009) (Fig. 2); therefore, al., 2009). Similarly, the C. elegans SLX4 orthologue HIM-18 is future experiments should address whether Mph1, like RTEL1, has required for processing meiotic recombination intermediates, a role in meiotic SDSA. because him-18 mutants display phenotypes that are consistent with reduced meiotic crossover formation (Saito et al., 2009). Possible mechanisms for crossover Moreover, HIM-18 physically interacts with the structure-specific interference endonucleases SLX-1 and XPF-1, and might serve as a scaffold The proteins discussed thus far all make an individual contribution that binds certain nucleases to allow cleavage of Holliday junction to the formation of either a crossover or non-crossover. These roles intermediates in various cellular contexts (Saito et al., 2009). For must be carefully regulated and work in concert for successful further details on recent advances in the area of resolvases, the completion of either crossover or non-crossover repair. However, reader is directed elsewhere (Mimitou and Symington, 2009; the factors described above are not the only factors that control the Svendsen and Harper, 2010). decision between crossover and non-crossover; it is also influenced by crossover interference, assurance and homeostasis on a Anti-crossover proteins chromosome-wide and genome-wide scale. Several proteins are known to inhibit crossover formation by It is well known that crossovers are distributed non-randomly promoting different pathways for meiotic DSB repair, and these along chromosomes. This phenomenon exists because of two involve alternative processing of recombination intermediates. underlying factors: first, DSBs are not formed randomly and, second, BLM, together with topoisomerase TOPOIII, has double Holliday crossover choice is governed by crossover interference. Crossover junction dissolution activity in vitro, which resolves these junctions interference dictates that adjacent crossovers on the same without crossover formation and, thereby, explains the increased chromosome tend to occur at sites that are further apart than degree of sister chromatid exchange observed in BLM-deficient expected if they were randomly positioned. Crossover assurance cells (Wu and Hickson, 2003) (Figs 1 and 2). The budding yeast makes certain that all chromosomes will obtain at least one BLM homologue Sgs1 suppresses the formation of multi-chromatid crossover, known as the obligate crossover. The presence of at least joint molecules during meiosis and, thereby, prevents aberrant one crossover between homologous chromosomes serves as a crossing over (Oh et al., 2007). These anti-crossover activities of physical linkage between homologues so that they can each attach Sgs1 are normally antagonised by the ZMM proteins (Jessop et al., to the correct spindle and, subsequently, segregate accurately in 2006). Conversely, the fission yeast BLM homologue Rec Q meiosis I. In budding yeast, crossover interference and assurance helicase 1 (Rqh1) might extend hybrid DNA and, thereby, bias the are separately regulated by the ZMM proteins (Shinohara et al., recombination outcome toward crossover formation (Cromie et al., 2008). Mutations in the ZMM genes result in defective SC formation 2008). In C. elegans mutants for the BLM orthologue him-6, together with reduced crossovers, but non-crossovers are unaffected meiotic crossovers are decreased compared with those in wild type (Borner et al., 2004). Different subcomplexes formed by ZMM

Journal of Cell Science (Wicky et al., 2004). Similarly, meiotic recombination is reduced proteins might have different functions. For instance, Mer3 and the to about half of the wild type frequency in D. melanogaster with Msh4–Msh5 heterodimer participate in promoting the differentiation mutations in the BLM orthologue mus309 (McVey et al., 2007). of crossovers, which establishes crossover interference; whereas Therefore, BLM might have context-dependent effects or differing the newly identified ZMM protein Spo16 acts in complex with Zip4 roles in different organisms and, thus, could be classified as having to efficiently implement crossovers, which is important for crossover both pro- and anti-crossover activities. It is also possible that the assurance (Shinohara et al., 2008). However, observations by de pro-crossover function of BLM reflects a role in meiotic DSB Boer et al. (de Boer et al., 2007) suggest that, in mice, a separate resection (Fig. 2). mechanism for crossover assurance, in addition to that of The C. elegans anti-recombinase RTEL-1 promotes non- interference, is not necessary (de Boer et al., 2007). crossover repair both in meiosis and (Barber et al., 2008; C. elegans meiosis presents an extreme case for the control of Youds et al., 2010). Analysis of the meiotic phenotypes of rtel-1 crossover formation. Here, meiotic crossover interference is referred mutants revealed that these animals had multiple crossovers per to as ‘complete’ because each chromosome has only a single chromosome, whereas wild type animals usually have only a single crossover, indicating that the number of crossovers per chromosome crossover per chromosome (Youds et al., 2010). rtel-1 mutants also is tightly regulated (Hillers and Villeneuve, 2003; Wood, 1988); have an increased number of recombination foci, as marked by <1% of oocyte meioses have a chromosome that lacks a crossover ZHP-3 in meiotic nuclei, indicating excess crossovers. RTEL-1 (Dernburg et al., 1998) and double crossovers are very rare (Lim has the ability to disrupt D loop recombination intermediates in et al., 2008). Studies of this system have shown that complete vitro (Barber et al., 2008; Youds et al., 2010) (Fig. 2). Thus, RTEL- crossover interference in C. elegans can be altered by either 1 is likely to promote non-crossovers by functioning in meiotic increasing the number of meiotic DSBs, for example, by mutating SDSA, where it probably dissociates the D loop intermediate to the condensin dumpy 28 (dpy-28), or by changing the balance facilitate the association of the invading DNA strand back with the between crossover and non-crossover repair of meiotic DSBs (by original duplex DNA, thereby inhibiting a stable strand invasion mutation in rtel-1). It is, therefore, possible that crossover event. In addition to its meiotic functions, RTEL1 acts in interstrand interference is regulated on at least two levels in C. elegans: by the crosslink repair in C. elegans and human cells, and is required for number of DSBs generated and by the pathway through which maintenance of telomere length in mice (Barber et al., 2008; Ding they are repaired. et al., 2004). Thus, RTEL1 is believed to have important roles as Related to crossover assurance and interference is crossover an anti-recombinase in multiple cellular scenarios. BLM and homeostasis, a mechanism that regulates the balance between Meiotic crossover and non-crossover control 509

crossovers and non-crossovers. In S. cerevisiae, experiments have buckling that places one recombination intermediate into a position shown that crossover homeostasis exists. Specifically, a set level where it commits to becoming a crossover and, subsequently, of crossovers is always maintained at the expense of non- tension is released in the area near the crossover so that no other crossover pathways, such as SDSA, in situations where fewer DSBs nearby will form crossovers (Borner et al., 2004; Kleckner meiotic DSBs are generated (Chen et al., 2008; Martini et al., et al., 2004) (Fig. 4). 2006). In mutants with reduced Spo11 activity, which have Current evidence indicates that the establishment of crossover reduced numbers of meiotic breaks, crossover homeostasis is interference is linked to chromosome axis structure. In yeast, the maintained by shifting the ratio between crossovers and non- conserved ATPase pachytene checkpoint 2 (Pch2) regulates meiotic crossovers towards fewer non-crossovers (Martini et al., 2006). axis morphogenesis by controlling the overall levels and localisation Homeostasis also exists in C. elegans when additional breaks are patterns of the structural protein Hop1 and the association of Zip3 generated by ionising radiation (IR); when excess DSBs are with meiotic chromosome axes (Joshi et al., 2009), but it is not induced by IR treatment in wild type animals, little or no increase required for crossover formation (Joshi et al., 2009; Zanders and in recombination is observed (Youds et al., 2010). However, in Alani, 2009). A proposed model for Pch2 function is that it rtel-1 mutants treated with IR, a large, dose-dependent increase reorganises chromosome axes into long-range, one-crossover in recombination is observed, indicating that rtel-1 mutants cannot modules, in which only a single crossover is assured and additional maintain homeostasis in the presence of additional meiotic DSBs crossovers are prevented (Joshi et al., 2009). The authors suggest (Youds et al., 2010). Homeostasis and crossover interference are that multiple single-crossover modules exist along the length of potentially regulated by a common mechanism because mutants each homologous chromosome pair, and that tension release – as defective in interference, such as budding yeast zip2 and zip4 proposed by the stress model (Borner et al., 2004; Kleckner et al., mutants and C. elegans rtel-1 mutants, also show compromised 2004) – prevents multiple crossovers from occurring within any homeostasis (Chen et al., 2008; Joshi et al., 2009; Youds et al., one module (Joshi et al., 2009). Therefore, higher-order 2010). chromosome structure appears to have an important role in Several models have been proposed over the years to explain crossover interference, at least in yeast. how crossover interference is propagated (Fig. 4). One early Data from mouse studies have also provided insights into the hypothesis in the field is the polymerisation model, in which the establishment of crossover interference. It has been reported that completion of a crossover initiates polymerisation of an inhibitor MLH1 foci, which mark crossovers in meiotic pachytene in mice, of recombination along the chromosome (King and Mortimer, exhibit strong interference (de Boer et al., 2006). The same group 1990). More recently, the stress model has been proposed, which showed that, prior to pachytene, late zygotene MSH4 foci and RPA posits that mechanical stress along the chromosome results in axis foci – which mark interhomologue interactions that could be

Polymerisation model Stress model

Journal of Cell Science Compression stress on axis exists

Potential crossover sites designated

Fig. 4. Illustration of two proposed mechanisms Crossover formation Axis buckling occurs of crossover interference. In the polymerisation model (left), the formation of a crossover (blue X) leads to the localisation of a recombination inhibitor (orange oval) at the crossover and its polymerisation along the vicinity, preventing other potential sites (blue circle) nearby from forming Recombination inhibitor localisation crossovers, thereby allowing non-crossover repair Crossover formation allows local stress release (green region of DNA) instead. In the mechanical stress model (right), compression stress (red arrows) along the chromosome axis leads to buckling of the axis at one of the potential crossover sites. Buckling at the site changes the Recombination inhibitor polymerisation prevents crossover, configuration of the recombination intermediate, allows non-crossover repair Axis relaxation occurs within the crossover region; other potential crossovers are repaired as non-crossovers committing it to become a crossover. Crossover formation (blue X) at this site allows local stress release, preventing other crossovers from forming close by. Similar to the polymerisation model, other potential recombination sites nearby are repaired as non-crossovers (green region of DNA). 510 Journal of Cell Science 124 (4)

resolved into either crossovers or non-crossovers – also exhibit but many questions remain regarding these processes. Further substantial interference (de Boer et al., 2006). These data are details are needed to clarify how a higher-order chromosome consistent with the idea that interference occurs in at least two structure contributes to crossover interference and whether a role consecutive steps. Furthermore, mice that lack the axial element for the chromosome axis in interference is conserved in higher protein SYCP3 show structurally abnormal axial elements and organisms. If a common mechanism controls one or several of incomplete chromosome synapsis, but have similar levels of these processes, how does this involve the chromosome axis and crossover interference when compared with wild type animals, other factors? Mutants that display phenotypes in which interference indicating that axial element structure and full synapsis are not and homeostasis defects are uncoupled would be highly valuable required for the establishment of normal interference (de Boer et to address these questions. Whatever the answers to these questions, al., 2007). However, correlations between SC length and future investigations into the factors that control the crossover- interference in mammals have been reported by other groups (Lynn non-crossover decision promise to be intriguing. et al., 2002; Petkov et al., 2007), so further research will be required to clarify the exact relationship between the SC, meiotic We acknowledge Carrie Adelman for images of mouse MLH1 foci, chromosome structure and crossover interference. and thank Jennifer Svendsen and Carrie Adelman for comments on the One further possibility is that epigenetic signals are also involved manuscript. 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