The choice in meiosis – defining the factors that ...axis has an important role in meiotic DSB...

Commentary 501

IntroductionMeiosis is the specialised reductive division that generates haploidcells. During this process, a single round of replication is followedby two rounds of chromosome segregation: in the first division(meiosis I), homologous chromosomes segregate, whereas in thesecond division, sister chromatids segregate (meiosis II). A keystep in meiosis I is the recognition of homologous chromosomes,which then align and pair along the length of the chromosome.Once homologues have aligned, synapsis can proceed with theformation of the synaptonemal complex (SC), a protein structurethat supports and maintains homologues in close juxtaposition andserves as a scaffold for crossover-promoting recombination factors.Meiotic crossing over involves the generation of meiotic double-strand breaks (DSBs), which are subsequently repaired either ascrossovers or non-crossovers (Fig. 1). Meiotic recombination isnot only necessary to create new allele combinations that generategenetic diversity, but is also essential in ensuring accuratechromosome segregation at the first meiotic division because thecrossover acts as a tether between homologues, which ensures thateach homologue will properly align at the metaphase plate andthereby correctly attach to the spindle. DSB repair occursconcurrently with SC formation and is required for normal synapsisin yeast and mice (Baudat et al., 2000; Roeder, 1997; Romanienkoand Camerini-Otero, 2000), whereas in Caenorhabditis elegansand Drosophila melanogaster, homologue pairing and SC formationcan occur independently of meiotic recombination (Colaiacovo etal., 2003; Dernburg et al., 1998; Liu et al., 2002; McKim et al.,1998).

The process of meiotic recombination is initiated when meioticDSBs are created by the endonuclease SPO11, in conjunction witha number of additional proteins (Keeney and Neale, 2006). DSBsare then resected to generate 3� single-strand DNA (ssDNA)

overhangs that are initially bound by replication protein A (RPA),which is subsequently displaced by the recombinase radiationsensitive 51 (RAD51) and/or the meiosis-specific recombinasedosage suppressor of Mck1 (DMC1) to form nucleoproteinfilaments. These filaments serve to find a complimentary sequencewithin a homologous chromosome, at which they instigate single-end strand invasions (Hunter and Kleckner, 2001) to generate so-called displacement loop (D loop) recombination intermediates(Fig. 1). If the second end of the original DSB binds with thehomologous chromosome, a double Holliday junction is formed,which can be resolved to generate either a non-crossover or aninterhomologue crossover, the latter of which is hereafter referredto simply as crossover (Bishop and Zickler, 2004; Schwacha andKleckner, 1995). Double Holliday junctions can also be processedthrough dissolution, which results in a non-crossover (Fig. 1) (Wuand Hickson, 2003). Meiotic non-crossovers have also beenproposed to form when strand invasion is transient, and when alimited amount of DNA synthesis occurs before the invaded stranddissociates and anneals to its partner strand, as in mitotic synthesis-dependent strand annealing (SDSA; see Box 1 and Fig. 1) (Allersand Lichten, 2001; Bishop and Zickler, 2004). During meiosis inbudding yeast, non-crossover heteroduplex products are found toform with the same timing as double Holliday junctions, whereascrossovers occur later (Allers and Lichten, 2001), consistent withthe idea that crossovers and non-crossovers are formed throughdistinct pathways. Analysis of ZMM mutants (Zip1, Zip2, Zip3,Zip4, Mer3, Msh4 and Msh5; further discussed below) in yeastindicates that the decision between crossover and non-crossover ismade very early, i.e. at or prior to the establishment of a stablesingle-end invasion intermediate, when one of the two ends of theDSB invades its homologous chromatid (Bishop and Zickler, 2004;Borner et al., 2004; Hunter and Kleckner, 2001). Thus, the

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

Key words: DSB repair, Meiosis, Crossover control, Homologous recombination

Journal of Cell Science 124, 501-513 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.074427

The choice in meiosis – defining the factors thatinfluence crossover or non-crossover formationJillian 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])

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crossover–non-crossover decision is thought to occur around thetime of strand exchange.

This Commentary will discuss the factors that contribute tocrossover or non-crossover formation in meiosis, including thegeneration and positioning of meiotic DSBs, formation of the SCand generation of recombination intermediates. We will also discusshow interhomologue crossing over is promoted compared withintersister repair, how recombination intermediates are resolvedinto crossovers as well as how anti-recombinases prevent crossingover. Crossover interference, assurance and homeostasis will alsobe discussed (see text box), including a summary of the currentmodels for how crossover interference is established.

Control of meiotic DSB formationMeiotic crossovers tend to form at specific sites in the genome ofmost organisms (Buard and de Massy, 2007; Mezard, 2006; Pryceand McFarlane, 2009); these are known as recombination hotspots.In mammals, hotspots are typically regions of 1–2 kb that occurevery 50–100 kb within the genome (Jeffreys et al., 2001; McVean,2010; Myers et al., 2008). Human recombination hotspots oftenoccur relatively close to genes (within 50 kb), but are preferentiallyfound outside of transcribed regions (Myers et al., 2005). Athotspots in eukaryotes, meiotic DSBs are generated by theconserved topoisomerase-like endonuclease Spo11 (Bergerat et al.,1997; Cao et al., 1990; Keeney et al., 1997). However, insightsinto what controls the position of break formation were unknownuntil recently.

Several studies have shown that Spo11 access to DNA is one ofthe determinants of DSB location, because DSBs are often foundwithin open chromatin in yeast (Berchowitz et al., 2009; Ohta etal., 1994; Wu and Lichten, 1994). In C. elegans, the putativechromatin-modifying protein high incidence of males 17 (HIM-17) is required for successful DSB formation and for normal levelsof dimethylation of lysine residue 9 of histone 3 (H3K9Me2)during meiosis, because mutants display a marked reduction inH3K9Me2 staining (Reddy and Villeneuve, 2004). Furthermore,DSB formation in him-17 non-null mutants can be enhanced byloss of LIN-35, the C. elegans retinoblastoma (Rb) proteinhomologue, which is a known component of chromatin modifyingcomplexes (Reddy and Villeneuve, 2004). Collectively, these datasuggest that the activity of HIM-17 alters the chromatin state,probably by opening the chromatin to allow access by SPO-11,and this governs DSB competence. More recently, components ofthe C. elegans condensin I complex have been implicated inregulating both the position and number of DSBs, because mutantsshow increased formation of meiotic DSBs and a distribution ofcrossovers that differs from the wild type (Mets and Meyer, 2009;Tsai et al., 2008). Here, it was demonstrated that disruption of thecondensin I complex causes an increase in chromosomal axislength, which is independent of DSB formation but requires theaxis-associated protein HIM-3 (Mets and Meyer, 2009). Thus, inC. elegans, it appears that the condensin I complex controlschromosome structure and the association of chromatin with thechromosome axis, which in turn might determine its accessibility

502 Journal of Cell Science 124 (4)

Double Holliday junction

Single-end invasion

Crossover Non-crossover

Double-strand-break formation

Non-crossover

or

Non-crossover

SDSA Double Hollidayjunction dissolution

Double Holliday junction resolution

D loop recombination intermediate

5�3�

5�3�

3�5�

Crossover (interference-dependent) (interference-independent)

Fig. 1. Model for meiotic crossover or non-crossoverformation. Double strand breaks are generated and their5� ends are resected to generate a 3� overhang. A strandinvasion event then generates a single-end invasion Dloop intermediate. If the second end of the original DSBalso engages with the homologue, a double Hollidayjunction is formed (shown on the left). The doubleHolliday junction can be resolved to form either acrossover (interference-dependent) or a non-crossover.Alternatively, the junction can be dissolved by doubleHolliday junction dissolution to form a non-crossover.Instead of forming a double Holliday junction, the Dloop can be dissociated and the invading strand canassociate with the opposite end of the original break, asin synthesis-dependent strand annealing (SDSA), to forma non-crossover. Alternatively, the intermediate can beacted upon by enzymes such as Mus81 that can forminterference-independent crossovers.

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by SPO-11. This and other evidence suggests that the chromosomeaxis has an important role in meiotic DSB formation. Certainly,proteins associated with the chromosome axis have also beenshown to be important for DSB formation. For example, the C.elegans HIM-3 paralog – him three paralog 3 (HTP-3) – is acomponent of the meiotic chromosome axis that mediates DSBformation through its interaction with a complex required togenerate DSBs (Goodyer et al., 2008). The conserved mouseprotein meiosis-specific 4 (MEI4), which associates with the axisof meiotic chromosomes, was also shown to be required for DSBformation (Kumar et al., 2010). Thus, a functional relationshipappears to exist between the chromosome axis and DSB formationand might have some bearing on where hotspots arise. For adetailed discussion and models of the interplay between thechromosome axis and DSB formation, readers are referred to thearticle by Kleckner (Kleckner, 2006).

If DSB hotspots are associated with sites of open chromatin,then the question arises what exactly determines the chromatinstate at hotspots. Evidence suggests that some recombinationhotspots in humans are associated with specific sequence motifs.Ten percent of the hotspots identified in a genome-wide survey of~1.6 million single-nucleotide polymorphisms (SNPs) in threesample populations were associated with the 7-mer motifCCTCCCT (Myers et al., 2005). Using more detailed data fromthe human ‘haplotype map’ (HapMap) project, work by thesame group identified a degenerate 13-mer sequence(CCNCCNTNNCCNC) that was present in one or more copies at40% of all human hotspots (Myers et al., 2008). These data indicatethat there is some correlation between DNA sequence and hotspotlocation. However, although humans and chimpanzees have morethan 98% sequence identity, hotspot locations in these two speciesare not conserved (Ptak et al., 2005; Winckler et al., 2005). Inaddition, there is evidence that recombination rates vary bothbetween ethnic groups (Evans and Cardon, 2005) and betweenindividuals (Cheung et al., 2007; Coop et al., 2008). Thus, sequencealone is clearly not the only factor determining hotspot activity.

Recent studies indicate a role for specific epigeneticmodifications at DSB sites. A recent study in C. elegans identifiedthe new chromatin factor X-non-disjunction factor 1 (XND-1),which is required for DSB formation specifically on the Xchromosome, as well as for the distribution of DSB formation (butnot DSB number) on the autosomes (Wagner et al., 2010). Increasedacetylation of histone H2A at lysine 5 (H2AK5Ac) that is likely tobe mediated by the histone acetylation protein Myst family histoneacetyltransferase-like (MYS-1; TIP60 in humans) in xnd-1 mutantswas identified as a chromatin modification associated with thealteration in autosomal distribution of meiotic DSBs and reducedDSB formation on the X chromosome (Wagner et al., 2010). Thisstudy suggests that H2AK5Ac is a determinant of DSB distributionin C. elegans, although the influence of this modification on DSBformation in other organisms still needs to be shown. Trimethylationof lysine 4 of histone 3 (H3K4Me3) has been considered as a pre-existing marker for sites of DSB formation in S. cerevisiae becausethis histone modification is frequently found in regions close toDSB sites, independently of gene 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 H3K4methyltransferase in S. cerevisiae, display a dramatic reduction inDSBs, and that those DSBs that form in the absence of Set1 aredifferentially localised (Borde et al., 2009). Specific histonemodifications were also shown to be present at meiotic DSB

initiation sites in mice with H3K4Me3 enriched at active DSBsites, whereas histone H4 hyperacetylation was found to occurafter DSB formation (Buard et al., 2009). H3K4Me3 is present inSpo11–/– mice, which lack meiotic DSBs, and thus H3K4Me3 is amarker for sites of DSB initiation that might contribute to hotspot

503Meiotic crossover and non-crossover control

Box 1. Terms used to describe meiosisAnti-recombinase: A protein that acts to prevent or inhibitrecombination.Chiasmata: The (cytologically) visible structure that is thephysical manifestation of a crossover between homologouschromosomes in meiosis.Crossover assurance: The mechanism that makes certain eachhomologous chromosome pair will receive at least one meioticcrossover.Crossover homeostasis: A mechanism that ensures a constantnumber of meiotic crossovers, even under conditions where moreor fewer meiotic DSBs are generated. Homeostasis maintainsmeiotic crossovers at the expense of non-crossovers. Howhomeostasis is maintained is not well understood but it might begoverned by the same mechanism as interference.Crossover interference: A mechanism that distributes meioticDSBs and crossovers such that adjacent crossovers tend tooccur further apart than expected by chance. The basis ofcrossover interference is not well understood.D loop: A recombination intermediate structure wherein one endof the DSB has invaded into the homologous chromosome andbeen extended to form a stable structure. A D loop can bedissociated to allow non-crossover repair through SDSA, or cango on to form a double Holliday junction. See Fig. 1.Double Holliday junction: A recombination intermediatestructure that can be formed following D loop formation if thesecond end of the original DSB also associates with thehomologous chromosome. This structure can be processed intoeither a crossover or non-crossover. See Fig. 1.Haplotype map: A map generated to describe common patternsof human genetic variation using single nucleotidepolymorphisms in various populations. Also known as theHapMap.Interference-independent crossovers: Crossovers that do notexhibit interference. Crossovers in this class are formed byMus81 catalyzed recombination intermediate cleavage in manyorganisms. Because they do not display interference,interference-independent crossovers can occur in close proximity.Interference-independent crossovers contrast with interference-dependent crossovers, which always display interference in termsof their positioning.Interhomolog crossover: Also known as a meiotic crossover, acrossover that has occurred between homologous chromosomesduring meiosis.Intersister repair: Repair of damage or a meiotic DSB (as in thisarticle) that uses the sister chromatid as a template rather thanthe homologous chromosome. Repair between sister chromatidscan be through crossover or non-crossover pathways.Synthesis-dependent strand annealing (SDSA): A pathway fornon-crossover repair wherein a D loop recombinationintermediate is formed and limited DNA synthesis occurs in theregion of the break, using the homologous chromosome as atemplate. The D loop is then dissociated and the invading end ofthe DSB that has been extended anneals back with the other endof the original DSB. DNA synthesis and ligation seals the break.ZMM proteins: A group of proteins that includes Zip1, Zip2, Zip3,Zip4, Mer3, Msh4, Msh5 and the recently identified Spo16 in S.cerevisiae; they are involved in both SC formation and meioticcrossing over.

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activity (Buard et al., 2009). HapMap-methylation-associated SNPs,which are markers of germline methylation, are positively correlatedwith regional meiotic recombination rates in humans (Sigurdssonet al., 2009), providing further evidence that epigeneticmodifications influence hotspot activity in mammals.

It remains unclear exactly how sites of frequent meioticrecombination are identified and marked as hotspots. However,recent studies in mice have identified the gene PR-domain-containing 9 (Prdm9) (Baudat et al., 2010; Parvanov et al., 2010),which encodes for a protein with histone methyltransferase activity.Different human alleles of PRDM9 show altered activity ofrecombination hotspots (Baudat et al., 2010). Furthermore, in vitro,the protein product of the human PRDM9 A allele was shown tobind to the specific 13-mer motif previously associated withrecombination hotspots, suggesting that PRDM9 marks DSBinitiation sites by recognising this motif (Baudat et al., 2010).Thus, sequence motifs that are modified by certain epigeneticmarks appear to designate some of the known mammalianrecombination hotspots by either signalling to the recombinationmachinery or allowing SPO11 to access the DNA. However, it isnot known how these signals integrate with factors that control theassociation of chromatin with the chromosome axis. Furthermore,the H3K4Me3 mark is highly enriched in promoter regions ofactively transcribed genes (Bernstein et al., 2005; Schneider et al.,2004), but meiotic DSBs do not form in all promoter regions and,currently, there is no known direct relationship between DSBformation and transcriptional activity (Hunter, 2006; Kniewel andKeeney, 2009). Thus, this chromatin modification alone is not

likely to account for all hotspot activity. As the roles of epigeneticmodifications in meiotic DSB formation and crossover controlremain poorly understood, they should be an important focus offuture investigation.

Proteins that influence crossover outcomesThe factors influencing DSB position are not yet well-understood,but a number of proteins that function downstream of DSB creation,to either promote or prevent crossover formation, have beencharacterised. Although it is not known exactly how the decisionis made to form a crossover or non-crossover, many meioticproteins influence whether or not a crossover can take place. Here,these proteins will be classified as having either pro-crossover oranti-crossover activities, although at least one of these proteins canbe considered to fit into both categories, depending on the contextof the protein activity or the model system in which it has beenexamined. A non-exhaustive list of these genes is presented inTable 1.

Pro-crossover proteinsSetting the stage for crossover formationA number of pro-crossover proteins have roles in pairing or SCformation that indirectly promote crossovers. SC formation involvesthe assembly of several structural elements, specifically a pair oflateral elements connected by transverse elements and a centralelement, which facilitates crossovers by continuously maintaininghomologues in close juxtaposition (Costa and Cooke, 2007; deCarvalho and Colaiacovo, 2006). Among the proteins that form the


Table 1. A non-exhaustive list of genes with either pro- or anti-crossover activitiesS. pombe S. cerevisiae C. elegans Drosophila Arabidopsis Vertebrates

Pro-crossover activityDSB end resection rad32, rad50,

nbs1 (complex)mre11, rad50,xrs2 (complex)

mre-11, rad-50 mre11, rad50,nbs

Atmre11,Atrad50, Atnbs1

MRE11, RAD50, NBS1(complex)a

ctIP sae2 com-1 CG5872?a Atgr1/Atcom1 CtIPa

rqh1 sgs1 him-6 dmBlm Atrecq4a BLMa

exo1 exo1 exo-1 tos ? EXO1a

dna2 dna2 dna-2 ? ? DNA2a

rhp51 rad51 rad-51 spnB Atrad51 RAD51a

dmc1 dmc1 absent absent Atdmc1 DMC1Crossover intermediate

formationrad22 rad52 brc-2 brca2 Atbrca2 BRCA2a

mer3 Atmer3/rckrhp54 rad54 rad-54 rad54 Atrad54 RAD54a

absent msh4, msh5 msh-4/him-14,msh5

absent Atmsh4, Atmsh5 MSH4, MSH5

mlh1 mlh1 mlh-1a mlh1 Atmlh1 MLH1mlh3 Atmlh3 MLH3

hop1 hop1rad3a mec1 atl-1a mei-41 Atatra ATRa

Promote interhomologcrossing over

rad3a tel1 atm-1a atm/tefua Atatma ATM a

mek1 mek1rad50a rad50a rad-50 rad50a Atrad50a RAD50a

mus81, eme1 mus81, mms4 mus-81, eme-1 mus81, mms4 Atmus81 MUS81, EME1

rad16b rad1b him-9 mei-9 Atrad1b XPF b

Crossover intermediateprocessing (complex) (complex) (complex) (complex) (complex)

(complex) (complex) (complex) (complex) (complex)

absent yen1 gen-1b gen1a ? GEN1a

slx1, slx4 slx1, slx4 slx-1, him-18 slx-1, mus312 ? SLX1, SLX4/BTBD12a

Anti-crossover activitydHJ 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.

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SC in S. cerevisiae are the Zip proteins (part of the ZMM group ofproteins). Zip1 is a structural protein that makes up the centralregions of the SC (Sym et al., 1993). Zip2, Zip3 and Zip4 are alsorequired for SC assembly, and have been implicated inubiquitylation and/or sumoylation of proteins associated with SCformation (Agarwal and Roeder, 2000; Borner et al., 2004; Chenget al., 2006; Chua and Roeder, 1998; Perry et al., 2005; Tsubouchiet al., 2006). A common theme among SC proteins is that, althoughthe sequence homologues of lower-organism SC proteins are noteasily detectible in higher organisms, structural features of theproteins are conserved (Costa and Cooke, 2007). In mammals, theSC is made up of multiple proteins including SC proteins 1, 2 and3 (SYCP1, 2 and 3), SC central element proteins 1 and 2 (SYCE1and SYCE2) and testis-expressed gene 12 (TEX12) (Costa andCooke, 2007), but these will not be discussed further here. Adetailed review of proteins that are involved in homologue pairingand synapsis, as well as recent insights into meiotic pairing centresin C. elegans that are beyond the scope of this review can be foundelsewhere (Costa and Cooke, 2007; Ding et al., 2010; Lynn et al.,2007; Yang and Wang, 2009; Zetka, 2009; Zickler, 2006).

Generation of the recombination intermediateIn the last few years, considerable progress has been made towardsdefining the activities that process DSBs to generate 3� ssDNAoverhangs, which are the substrate for initiating homologousrecombination. Studies of C. elegans completion of meioticrecombination-1 (com-1) and its homologue in Arabidopsis thaliana,com1 (homologues of yeast sae2, human CtIP), found a similarphenotype in these two species, in which mutants failed to loadRAD51 onto meiotic DSBs, suggesting that COM1 acts in DSBresection (Penkner et al., 2007; Uanschou et al., 2007). More recently,data from budding yeast and mammalian cells have revealed thatDSB resection is dependent on the cooperative action of multiplefactors, including the Mre11–Rad50–Xrs2 (MRX) complex [Mre11–Rad50–Nbs1 (MRN) in mammals] SUMO activating enzyme 2(Sae2; CtIP in mammals), small growth suppressor 1 [Sgs1; Bloom’ssyndrome mutated (BLM) in mammals], exonuclease 1 (Exo1;EXO1 in mammals) and DNA replication helicase 2 (Dna2; DNA2in mammals) (Gravel et al., 2008; Mimitou and Symington, 2008;Zhu et al., 2008) (Fig. 2). These studies have been validated by theanalysis of DSB end processing in vitro using the respective purifiedproteins (Nimonkar et al., 2008). This area has been intensivelyreviewed and discussed elsewhere and we refer the reader to severalreviews on this topic (Bernstein and Rothstein, 2009; Mimitou andSymington, 2009). Once the DSB has been processed, Rad51 and/orthe meiosis-specific recombinase Dmc1 bind the 3� ssDNA togenerate a nucleoprotein filament that carries out a homology searchand strand invasion into the homologous chromosome. Both Dmc1and Rad51 are required for efficient meiotic recombination in yeast(Schwacha and Kleckner, 1997) and mice (Pittman et al., 1998;Sharan et al., 2004; Yoshida et al., 1998), but Dmc1 orthologueshave not been identified in C. elegans or D. melanogaster. HumanDMC1 has robust D-loop-forming activity in vitro (Li et al., 1997;Sehorn et al., 2004), whereas in S. cerevisiae, Rad54 is requiredtogether with Rad51 for D loop formation (Sung et al., 2003). Invivo, the Dmc1 nucleoprotein filament is more adept at forming Dloops with the homologous chromosome than the Rad51nucleoprotein filament (Shinohara et al., 2003; Tsubouchi and Roeder,2003). Co-ordination of RAD51 and DMC1 activities in mammalianmeiosis might be achieved through the breast cancer susceptibilityprotein 2 (BRCA2), because BRCA2 binds to both proteins at

distinct sites (Sharan et al., 1997; Thorslund et al., 2007), and isrequired for localisation of RAD51 and DMC1 at foci that arepresumed to be meiotic DSBs in mice (Sharan et al., 2004). Similarly,C. elegans BRCA2 (BRC-2) is essential for meiotic DSB repair, inwhich it functions to promote RAD-51 filament nucleation andstabilisation, and in the stimulation of RAD-51 mediated strandexchange (Martin et al., 2005; Petalcorin et al., 2007; Petalcorin etal., 2006).

Mismatch repair defective 4 and 5 (Msh4 and Msh5, respectively)and meiotic recombination 3 (Mer3), i.e. other members of theZMMs, also promote crossovers, probably by facilitating the stableformation of recombination intermediates. msh4 and msh5 mutantsin S. cerevisiae have reduced inter-homologue crossing over(Hollingsworth et al., 1995; Ross-Macdonald and Roeder, 1994).Similarly, in C. elegans, MSH-4 and MSH-5 are essential forcrossing over (Kelly et al., 2000). Msh4 and Msh5 are known toform a heterodimer, and one proposal is that this acts as a clamp tohold homologous chromosomes together, thereby stabilising theHolliday junction and facilitating crossing over (Snowden et al.,2004) (Fig. 2). In Msh4 and Msh5 knockout mice, chromosomesfail to correctly pair, crossovers are absent and, consequently,animals are sterile (de Vries et al., 1999; Edelmann et al., 1999;Kneitz et al., 2000). In mammals, unlike in S. cerevisiae, Msh4 andMsh5 are required for correct chromosome pairing during meioticprophase, and both are clearly associated with recombinationintermediates destined to form both crossovers and noncrossovers(Kneitz et al., 2000; Santucci-Darmanin et al., 2000). Thus, themouse phenotype associated with loss of Msh4 or Msh5 does notonly reflect of a loss of crossovers, but is also more severe than thatof other mutants, such as loss of the mut L homologue 1 (Mlh1;discussed below) (Edelmann et al., 1996). Interestingly, msh4 andmsh5 are not found in D. melanogaster and in S. pombe. The latterrelies solely on the structure-specific endonuclease complex betweenmutagenesis sensitive 81 (Mus81) and essential meioticendonuclease 1 (Eme1) for crossover formation (Osman et al.,2003), whereas in D. melanogaster the clamp function of Msh4 andMsh5 can potentially be replaced by alternative proteins or analternative mechanism might be present (Blanton et al., 2005). Inaddition to Msh4 and Msh5, the Mer3 helicase promotes normalcrossover frequencies in yeast (Nakagawa and Ogawa, 1999). Mer3functions to stimulate heteroduplex extension by Rad51, therebystabilising nascent D loop structures to promote capture of thesecond free DNA end, double Holliday junction formation and,ultimately, crossovers (Mazina et al., 2004). In Sordaria, Mer3,Msh4 and Mlh1 have recently been shown to have roles inhomologous chromosome pairing (Storlazzi et al., 2010).

MLH1 is a well-known marker for sites designated as crossoversin the mouse (Anderson et al., 1999). MLH1 is required forcrossovers, as germ cells from mice that lack MLH1 do not displaysufficient chiasmata and do not progress beyond the meioticpachytene stage, and thus, Mlh1 null mice are infertile (Edelmannet al., 1996) (Fig. 3). Budding yeast mlh1 deletion mutants havereduced crossing over (Hunter and Borts, 1997). In vitro, humanMSH4 interacts with MLH1, and in vivo the two proteins colocaliseduring early to mid-pachytene, when crossing over takes place(Santucci-Darmanin et al., 2000). One possible function of MLH1is that it mediates release of the MSH4 and MSH5 clamp (Snowdenet al., 2004), thereby facilitating crossover completion. mlh-1 hasnot been characterised in C. elegans; instead, Zip homologousprotein 3 (ZHP-3), the C. elegans homologue of yeast Zip3, hasbeen shown to be a marker for meiotic crossovers (Fig. 3). ZHP-3


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has two roles in meiosis: it promotes crossover formation and alsorestructures chromosomes at diakinesis, the chromosomecondensation stage, so that appropriate segregation can take place(Bhalla et al., 2008; Jantsch et al., 2004). Thus, several proteins arerequired to generate and maintain a stable recombinationintermediate. In order for appropriate chromosome segregation totake place, the stable recombination intermediate must haveengaged the homologous duplex and not the sister chromatid,ultimately resulting in formation of an interhomologue crossover.

Promoting interhomologue crossing over versus intersisterrepairMeiotic crossover formation can be controlled by genes that allowmeiotic DSB repair from only certain templates, i.e. not all. Theproteins that participate in the barrier to sister chromatid repair arepro-crossover factors because they promote DSB repair that usesthe homologous chromosome, but not the sister chromatid (see textbox). In this way, these factors allow crossovers between

homologues but not between sister chromatids. For example, inbudding yeast, interhomologue recombination is ensured byphosphorylation of the axial element protein homologue pairing 1(Hop1) by the serine/theronine protein kinases meiotic checkpoint1 (Mec1) and telomere length 1 (Tel1), the homologues ofmammalian ataxia telangiectasia and Rad3-related protein (ATR)and ataxia telangiectasia mutated (ATM), respectively (Carballo etal., 2008). It has been proposed that Hop1 phosphorylation leadsto dimerisation of the meiotic axial element meiotic kinase 1(Mek1), which enables it to phosphorylate its target proteins thatprevent the repair of DSBs using the sister chromatid (Niu et al.,2005). In addition, Mek1 inhibits Rad51 activity by attenuating theformation of the Rad51–Rad54 complex; with less active Rad51,the activity of the meiosis-specific strand exchange protein Dmc1is favoured (Niu et al., 2009). Dmc1 is thought to be more efficientat promoting interhomologue recombination than Rad51 (Shinoharaet al., 2003; Tsubouchi and Roeder, 2003), thus, the activity ofMek1 promotes interhomologue recombination over sister


Enzyme

RTEL-1

Mph1

Activity

Strand displacement

Sgs1Rqh1HIM-6

MUS309BLM

Dissolution

DSB resection

Outcome

Non-crossover

?

Non-crossover

Pro-recombination

Yen1/GEN1

SLX4, HIM-18, MUS312, BTBD12

Resolution

Crossover and/ornon-crossover

Crossover

Crossover

Mus81

MEI9 (XPF) Strand cleavage

MSH4/5Pro-crossover

? MLH1/3

MER3Heteroduplex

extension

Pro-crossover

Helicases

Nucleases

DNA- bindingproteins

Fig. 2. Biochemical activities that promote crossover and/or non-crossover recombination. Shown are schematic representations of specific recombinationintermediates that are subject to biochemical activities of meiotic enzymes (helicases, nucleases, DNA-binding proteins) that act to promote one of two repairoutcomes: non-crossover or crossover (recombination). Arrows indicate the directionality of the helicase activity; arrows with scissors indicate the position ofcleavage of the nuclease 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 humanBTBD12, respectively.

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chromatid repair. In C. elegans – which do not have Dmc1 – germcells, at the onset of meiotic prophase, switch into a specialisedmode of DSB repair that is characterised by the requirement forRAD-50 in loading RAD-51 onto meiotic DSB ends, a processthat is essential for interhomologue crossover formation (Hayashiet al., 2007). By mid- to late pachytene, a second developmentallyprogrammed switch occurs; RAD-50 is no longer required forRAD-51 association with DSBs, and competence forinterhomologue crossing over is lost (Hayashi et al., 2007). Repairof any remaining DSBs might then occur through the sisterchromatid, which requires BRCA homologue 1 (BRC-1) that isdispensable for repair through the homologue (Adamo et al., 2008).The requirement for RAD-50 in RAD-51 loading at DSBs ispartially dependent on a number of proteins that have roles inmeiosis-specific chromosome axis structure (Hayashi et al., 2007).Thus, mechanisms exist to ensure that crossing over with thehomologue will occur at the correct time, rather than with the sisterchromatid. Once the stable interhomologue crossover intermediatehas been generated, the activity of a resolution enzyme willultimately complete the crossover.

Resolution of the recombination intermediateCrossover formation is controlled directly by enzymes that act onrecombination intermediates and resolve these either as crossoversor non-crossovers. Mus81–Eme1 can resolve intermediates intocrossovers (Fig. 2), but also has multiple biochemical abilities,including a preference for acting on structures that include Dloops, nicked Holliday junctions, replication forks with the laggingstrand at the junction point, and 3� flap structures (Bastin-Shanoweret al., 2003; Fricke et al., 2005; Osman et al., 2003). Yeast Mus81–Eme1 is also reported to have robust cleavage activity on intactHolliday junctions, although they are not its preferred substrate invitro (Gaskell et al., 2007). Mus81 is responsible for generatinginterference-independent crossovers in S. cerevisiae (Argueso etal., 2004; de los Santos et al., 2003) and in mouse (Holloway etal., 2008), and also generates the majority of crossovers in S.pombe, which has only interference-independent crossovers (Boddyet al., 2001; Osman et al., 2003; Smith et al., 2003). In C. elegans,MUS-81 is also responsible for a subset of crossovers that occurwhen meiotic non-crossover repair through SDSA is blocked bymutation in the anti-recombinase regulator of telomere length 1(RTEL-1) (Youds et al., 2010).

A number of DNA processing enzymes have recently beenidentified in various organisms that possess the biochemical activityto resolve Holliday junctions – specifically, the ability to cleaveHolliday junctions symmetrically. These resolvases have thepotential to generate either crossover or non-crossover products,and other factors, perhaps the MSH4-MSH5 complex, mightinfluence this decision. The human resolvase XPG-likeendonuclease 1 (GEN1), and its S. cerevisiae orthologue Yen1,have been independently identified through their ability tosymmetrically cleave Holliday junctions (Ip et al., 2008) (Fig. 2).Yen1 has functional overlap with Mus81 in budding yeast (Blancoet al., 2010) and human GEN1 was found to rescue the meioticphenotype of mus81 S. pombe mutants (Lorenz et al., 2009),indicating conserved, functionally similar roles for Mus81 andYen1 or GEN1 in yeast and humans, respectively. However, wehave not observed a meiotic phenotype for gen-1 mutants in C.elegans (J.L.Y. and S.J.B., unpublished data) and, together with therecently demonstrated role for gen-1 in DNA damage signallingand repair in the nematode (Bailly et al., 2010), this indicates thatadditional enzyme(s) act as the meiotic resolvase in this organism.

In addition to GEN1, concurrent works from several groupsdescribed roles for the orthologous proteins Drosophila Mus312,C. elegans HIM-18, and mammalian synthetic lethal of unknownfunction 4 (SLX4) in resolving Holliday junctions (Andersen etal., 2009; Fekairi et al., 2009; Munoz et al., 2009; Saito et al.,2009; Svendsen et al., 2009) (Fig. 2). In mus312 mutants of D.melanogaster, meiotic crossing over is reduced by ~95%, consistentwith the hypothesis that Mus312 is the key protein required forHolliday junction resolution in this organism (Andersen et al.,2009; Yildiz et al., 2002). Gene expression patterns and knockdownstudies suggest that mammalian SLX4 has a meiotic function inhumans that is conserved with that of D. melanogaster Mus312(Andersen et al., 2009). SLX4 interacts with SLX1, and the SLX4–SLX1 complex has a robust Holliday junction cleavage activity invitro, which probably accounts for its role in meiosis (Fekairi etal., 2009; Munoz et al., 2009; Svendsen et al., 2009). SLX4 alsobinds the structure-specific complex between the endonucleasesXeroderma Pigmentosum complementation group F and excisionrepair cross-complementing 1 (XPF–ERCC1) complex andMUS81–EME1, among other proteins. Depletion of SLX4 also


Wild type

rtel-1 mutant

DAPI + GFP GFP only

C. elegans crossover sites marked by ZHP-3

A

B

Mouse crossover sites marked by MLH1

SCP3 MLH1

Fig. 3. Examples of crossover interference. (A)Examples of crossoversmarked by staining of MLH1 (green) on mouse chromosomes. The SC ismarked in red by staining of the SC protein 3 (SCP3). When multiplecrossovers occur on the same chromosome, they tend to be spaced far apart,demonstrating crossover interference. (B)Complete crossover interference inC. elegans. The images in the top row show the single recombination foci perchromosome marked by staining of ZHP-3 (six chromosomes, hence six foci)at meiotic diplotene in a wild type animal. The rtel-1 mutant exhibits defectivecomplete crossover interference, as illustrated by the increased number ofZHP-3 foci per meiotic nucleus (bottom images).

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causes sensitivity to a number of DNA damage agents, indicatingthat it serves as a platform for different structure-specificendonucleases that are most likely to act both in meiosis and inDNA repair (Fekairi et al., 2009; Munoz et al., 2009; Svendsen etal., 2009). Similarly, the C. elegans SLX4 orthologue HIM-18 isrequired for processing meiotic recombination intermediates,because him-18 mutants display phenotypes that are consistentwith reduced meiotic crossover formation (Saito et al., 2009).Moreover, HIM-18 physically interacts with the structure-specificendonucleases SLX-1 and XPF-1, and might serve as a scaffoldthat binds certain nucleases to allow cleavage of Holliday junctionintermediates in various cellular contexts (Saito et al., 2009). Forfurther details on recent advances in the area of resolvases, thereader is directed elsewhere (Mimitou and Symington, 2009;Svendsen and Harper, 2010).

Anti-crossover proteinsSeveral proteins are known to inhibit crossover formation bypromoting different pathways for meiotic DSB repair, and theseinvolve alternative processing of recombination intermediates.BLM, together with topoisomerase TOPOIII, has double Hollidayjunction dissolution activity in vitro, which resolves these junctionswithout crossover formation and, thereby, explains the increaseddegree of sister chromatid exchange observed in BLM-deficientcells (Wu and Hickson, 2003) (Figs 1 and 2). The budding yeastBLM homologue Sgs1 suppresses the formation of multi-chromatidjoint molecules during meiosis and, thereby, prevents aberrantcrossing over (Oh et al., 2007). These anti-crossover activities ofSgs1 are normally antagonised by the ZMM proteins (Jessop et al.,2006). Conversely, the fission yeast BLM homologue Rec Qhelicase 1 (Rqh1) might extend hybrid DNA and, thereby, bias therecombination outcome toward crossover formation (Cromie et al.,2008). In C. elegans mutants for the BLM orthologue him-6,meiotic crossovers are decreased compared with those in wild type(Wicky et al., 2004). Similarly, meiotic recombination is reducedto about half of the wild type frequency in D. melanogaster withmutations in the BLM orthologue mus309 (McVey et al., 2007).Therefore, BLM might have context-dependent effects or differingroles in different organisms and, thus, could be classified as havingboth pro- and anti-crossover activities. It is also possible that thepro-crossover function of BLM reflects a role in meiotic DSBresection (Fig. 2).

The C. elegans anti-recombinase RTEL-1 promotes non-crossover repair both in meiosis and mitosis (Barber et al., 2008;Youds et al., 2010). Analysis of the meiotic phenotypes of rtel-1mutants revealed that these animals had multiple crossovers perchromosome, whereas wild type animals usually have only a singlecrossover per chromosome (Youds et al., 2010). rtel-1 mutants alsohave an increased number of recombination foci, as marked byZHP-3 in meiotic nuclei, indicating excess crossovers. RTEL-1has the ability to disrupt D loop recombination intermediates invitro (Barber et al., 2008; Youds et al., 2010) (Fig. 2). Thus, RTEL-1 is likely to promote non-crossovers by functioning in meioticSDSA, where it probably dissociates the D loop intermediate tofacilitate the association of the invading DNA strand back with theoriginal duplex DNA, thereby inhibiting a stable strand invasionevent. In addition to its meiotic functions, RTEL1 acts in interstrandcrosslink repair in C. elegans and human cells, and is required formaintenance of telomere length in mice (Barber et al., 2008; Dinget al., 2004). Thus, RTEL1 is believed to have important roles asan anti-recombinase in multiple cellular scenarios. BLM and

RTEL1 are the only examples of meiotic anti-crossover proteins inthe current literature. However, it has been reported that S.cerevisiae mutator phenotype 1 (Mph1) has biochemical abilitiessimilar to those of RTEL1 (Prakash et al., 2009) (Fig. 2); therefore,future experiments should address whether Mph1, like RTEL1, hasa role in meiotic SDSA.

Possible mechanisms for crossoverinterferenceThe proteins discussed thus far all make an individual contributionto the formation of either a crossover or non-crossover. These rolesmust be carefully regulated and work in concert for successfulcompletion of either crossover or non-crossover repair. However,the factors described above are not the only factors that control thedecision between crossover and non-crossover; it is also influencedby crossover interference, assurance and homeostasis on achromosome-wide and genome-wide scale.

It is well known that crossovers are distributed non-randomlyalong chromosomes. This phenomenon exists because of twounderlying factors: first, DSBs are not formed randomly and, second,crossover choice is governed by crossover interference. Crossoverinterference dictates that adjacent crossovers on the samechromosome tend to occur at sites that are further apart thanexpected if they were randomly positioned. Crossover assurancemakes certain that all chromosomes will obtain at least onecrossover, known as the obligate crossover. The presence of at leastone crossover between homologous chromosomes serves as aphysical linkage between homologues so that they can each attachto the correct spindle and, subsequently, segregate accurately inmeiosis I. In budding yeast, crossover interference and assuranceare separately regulated by the ZMM proteins (Shinohara et al.,2008). Mutations in the ZMM genes result in defective SC formationtogether with reduced crossovers, but non-crossovers are unaffected(Borner et al., 2004). Different subcomplexes formed by ZMMproteins might have different functions. For instance, Mer3 and theMsh4–Msh5 heterodimer participate in promoting the differentiationof crossovers, which establishes crossover interference; whereasthe newly identified ZMM protein Spo16 acts in complex with Zip4to efficiently implement crossovers, which is important for crossoverassurance (Shinohara et al., 2008). However, observations by deBoer et al. (de Boer et al., 2007) suggest that, in mice, a separatemechanism for crossover assurance, in addition to that ofinterference, is not necessary (de Boer et al., 2007).

C. elegans meiosis presents an extreme case for the control ofcrossover formation. Here, meiotic crossover interference is referredto as ‘complete’ because each chromosome has only a singlecrossover, indicating that the number of crossovers per chromosomeis tightly regulated (Hillers and Villeneuve, 2003; Wood, 1988);<1% of oocyte meioses have a chromosome that lacks a crossover(Dernburg et al., 1998) and double crossovers are very rare (Limet al., 2008). Studies of this system have shown that completecrossover interference in C. elegans can be altered by eitherincreasing the number of meiotic DSBs, for example, by mutatingthe condensin dumpy 28 (dpy-28), or by changing the balancebetween crossover and non-crossover repair of meiotic DSBs (bymutation in rtel-1). It is, therefore, possible that crossoverinterference is regulated on at least two levels in C. elegans: by thenumber of DSBs generated and by the pathway through whichthey are repaired.

Related to crossover assurance and interference is crossoverhomeostasis, a mechanism that regulates the balance between


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crossovers and non-crossovers. In S. cerevisiae, experiments haveshown that crossover homeostasis exists. Specifically, a set levelof crossovers is always maintained at the expense of non-crossover pathways, such as SDSA, in situations where fewermeiotic DSBs are generated (Chen et al., 2008; Martini et al.,2006). In mutants with reduced Spo11 activity, which havereduced numbers of meiotic breaks, crossover homeostasis ismaintained by shifting the ratio between crossovers and non-crossovers towards fewer non-crossovers (Martini et al., 2006).Homeostasis also exists in C. elegans when additional breaks aregenerated by ionising radiation (IR); when excess DSBs areinduced by IR treatment in wild type animals, little or no increasein recombination is observed (Youds et al., 2010). However, inrtel-1 mutants treated with IR, a large, dose-dependent increasein recombination is observed, indicating that rtel-1 mutants cannotmaintain homeostasis in the presence of additional meiotic DSBs(Youds et al., 2010). Homeostasis and crossover interference arepotentially regulated by a common mechanism because mutantsdefective in interference, such as budding yeast zip2 and zip4mutants and C. elegans rtel-1 mutants, also show compromisedhomeostasis (Chen et al., 2008; Joshi et al., 2009; Youds et al.,2010).

Several models have been proposed over the years to explainhow crossover interference is propagated (Fig. 4). One earlyhypothesis in the field is the polymerisation model, in which thecompletion of a crossover initiates polymerisation of an inhibitorof recombination along the chromosome (King and Mortimer,1990). More recently, the stress model has been proposed, whichposits that mechanical stress along the chromosome results in axis

buckling that places one recombination intermediate into a positionwhere it commits to becoming a crossover and, subsequently,tension is released in the area near the crossover so that no otherDSBs nearby will form crossovers (Borner et al., 2004; Kleckneret al., 2004) (Fig. 4).

Current evidence indicates that the establishment of crossoverinterference is linked to chromosome axis structure. In yeast, theconserved ATPase pachytene checkpoint 2 (Pch2) regulates meioticaxis morphogenesis by controlling the overall levels and localisationpatterns of the structural protein Hop1 and the association of Zip3with meiotic chromosome axes (Joshi et al., 2009), but it is notrequired for crossover formation (Joshi et al., 2009; Zanders andAlani, 2009). A proposed model for Pch2 function is that itreorganises chromosome axes into long-range, one-crossovermodules, in which only a single crossover is assured and additionalcrossovers are prevented (Joshi et al., 2009). The authors suggestthat multiple single-crossover modules exist along the length ofeach homologous chromosome pair, and that tension release – asproposed by the stress model (Borner et al., 2004; Kleckner et al.,2004) – prevents multiple crossovers from occurring within anyone module (Joshi et al., 2009). Therefore, higher-orderchromosome structure appears to have an important role incrossover interference, at least in yeast.

Data from mouse studies have also provided insights into theestablishment of crossover interference. It has been reported thatMLH1 foci, which mark crossovers in meiotic pachytene in mice,exhibit strong interference (de Boer et al., 2006). The same groupshowed that, prior to pachytene, late zygotene MSH4 foci and RPAfoci – which mark interhomologue interactions that could be


Polymerisation model Stress model

Recombination inhibitor localisation

Recombination inhibitor polymerisation prevents crossover,allows non-crossover repair

Crossover formation

Potential crossover sites designated

Compression stress on axis exists

Axis buckling occurs

Crossover formation allows local stress release

Axis relaxation occurs within the crossover region; other potential crossovers are repaired as non-crossovers

Fig. 4. Illustration of two proposed mechanismsof crossover interference. In the polymerisationmodel (left), the formation of a crossover (blue X)leads to the localisation of a recombinationinhibitor (orange oval) at the crossover and itspolymerisation along the vicinity, preventing otherpotential sites (blue circle) nearby from formingcrossovers, thereby allowing non-crossover repair(green region of DNA) instead. In the mechanicalstress model (right), compression stress (redarrows) along the chromosome axis leads tobuckling of the axis at one of the potentialcrossover sites. Buckling at the site changes theconfiguration of the recombination intermediate,committing it to become a crossover. Crossoverformation (blue X) at this site allows local stressrelease, preventing other crossovers from formingclose by. Similar to the polymerisation model,other potential recombination sites nearby arerepaired as non-crossovers (green region of DNA).

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resolved into either crossovers or non-crossovers – also exhibitsubstantial interference (de Boer et al., 2006). These data areconsistent with the idea that interference occurs in at least twoconsecutive steps. Furthermore, mice that lack the axial elementprotein SYCP3 show structurally abnormal axial elements andincomplete chromosome synapsis, but have similar levels ofcrossover interference when compared with wild type animals,indicating that axial element structure and full synapsis are notrequired for the establishment of normal interference (de Boer etal., 2007). However, correlations between SC length andinterference in mammals have been reported by other groups (Lynnet al., 2002; Petkov et al., 2007), so further research will berequired to clarify the exact relationship between the SC, meioticchromosome structure and crossover interference.

One further possibility is that epigenetic signals are also involvedin marking the crossover site and preventing other crossovers fromoccurring nearby. The above mentioned study that showed thatHapMap methylation-associated SNPs are positively correlatedwith regions of meiotic recombination (Sigurdsson et al., 2009)suggests that either methylation is a marker for DSB sites or,alternatively, its occurrence after crossover formation inhibitsfurther crossovers, which serve as the signal for interference.Further studies will be essential to understand the relationshipbetween epigenetic marks, chromosome axis morphogenesis andthe crossover–non-crossover decision.

Future perspectivesAlthough recent studies have enhanced our understanding of thefactors that control meiotic crossing over, many questions remain.Further experiments are needed to clearly define how thechromosome axis and its association with DNA control the locationof meiotic DSBs. Understanding the timing of epigenetic markswill also be important in clarifying how these DNA modificationscontrol DSB initiation. For example, one question is whether thereare different modifications at crossover and non-crossover sites.Prdm9 has recently been identified as a histone modifying proteinthat might mark DSB initiation sites (Baudat et al., 2010), but arethere other proteins that act similarly? Answers to these and otherquestions will help to establish the involvement of epigeneticsignals in designating DSB and/or crossover sites.

The activities of a number of pro-crossover proteins are relativelywell understood. However, only a few meiotic non-crossoverproteins have been identified. Given that, in most eukaryotes, farmore meiotic DSBs occur compared with the number of crossoversthat will eventually be formed, meiotic non-crossover proteinshave an extremely important role in preventing excess crossovers.Thus, additional anti-crossover proteins probably exist but need tobe identified. Synthetic lethal screens in the genetic backgroundsof anti-crossover mutants, such as rtel-1 or BLM, might identifyadditional genes with overlapping functions. Furthermore, anyproteins that can act on D loop recombination intermediates shouldbe tested for roles in meiotic crossover control. In addition, othermodes of regulating the promotion of non-crossovers should alsobe considered, aside from a direct activity on recombinationintermediates. For instance, are there proteins that promote DSBrepair by using the sister chromatid rather than the homologouschromosome? Or is the activity of certain proteins that aid in theformation or resolution of stable recombination intermediatestemporally inhibited to favour non-crossovers?

Finally, recent years have seen strides toward a greaterunderstanding of crossover interference, assurance and homeostasis,

but many questions remain regarding these processes. Furtherdetails are needed to clarify how a higher-order chromosomestructure contributes to crossover interference and whether a rolefor the chromosome axis in interference is conserved in higherorganisms. If a common mechanism controls one or several ofthese processes, how does this involve the chromosome axis andother factors? Mutants that display phenotypes in which interferenceand homeostasis defects are uncoupled would be highly valuableto address these questions. Whatever the answers to these questions,future investigations into the factors that control the crossover-non-crossover decision promise to be intriguing.

We acknowledge Carrie Adelman for images of mouse MLH1 foci,and thank Jennifer Svendsen and Carrie Adelman for comments on themanuscript. Research in the lab of S.J.B. is funded by Cancer ResearchUK. S.J.B. is a Royal Society Wolfson Research Merit Award holder.

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