Initiation of meiotic recombination how and where Conservation and specificities among eukaryotes_DeMassy_2013

GE47CH24-deMassy ARI 29 October 2013 15:11

Initiation of MeioticRecombination: How andWhere? Conservation andSpecificities AmongEukaryotesBernard de MassyInstitute of Human Genetics, Centre National de la Recherche Scientifique, UPR1142,34396 Montpellier, France; email: [email protected]

Annu. Rev. Genet. 2013. 47:563–99

The Annual Review of Genetics is online atgenet.annualreviews.org

This article’s doi:10.1146/annurev-genet-110711-155423

Copyright c© 2013 by Annual Reviews.All rights reserved

Keywords

meiosis, DNA double-strand break, genome stability, crossover, sexualreproduction

Abstract

Meiotic recombination is essential for fertility in most sexually repro-ducing species. This process also creates new combinations of allelesand has important consequences for genome evolution. Meiotic re-combination is initiated by the formation of DNA double-strand breaks(DSBs), which are repaired by homologous recombination. DSBs arecatalyzed by the evolutionarily conserved SPO11 protein, assisted byseveral other factors. Some of them are absolutely required, whereasothers are needed only for full levels of DSB formation and may par-ticipate in the regulation of DSB timing and frequency as well as thecoordination between DSB formation and repair. The sites where DSBsoccur are not randomly distributed in the genome, and remarkably dis-tinct strategies have emerged to control their localization in differentspecies. Here, I review the recent advances in the components requiredfor DSB formation and localization in the various model organisms inwhich these studies have been performed.

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Homologouschromosomes(homologs): the twoparental chromosomespresent in a diploid cell

Sister chromatids:the two chromatidsthat result from around of DNAreplication

INTRODUCTIONIn the sexual reproductive cycle, diploid cellsare converted into haploid cells through meio-sis, in which a single round of DNA repli-cation is followed by two divisions. Duringthe first meiotic division, homologous chromo-somes (homologs) segregate, and in the seconddivision, sister chromatids segregate. Thefirst segregation, called reductional segrega-tion, faces a situation unique to meiotic cells,whereby the two homologs must identify eachother and segregate to the opposite poles. Thisinvolves a specific process taking place during

Crossing over

Initiation of meiotic recombination DNA double-strand break (DSB)

Sister chromatid exchangeInterhomolog, NCO

DSB repair by homologous recombination

Nonhomologous end joining Ectopic (nonallelic) recombination

Sister chromatids

Cohesin

Homologs

Interhomolog, CO

Figure 1Connecting homologs by recombination. Meiotic double-strand breaks (DSBs) are induced at the beginningof the meiotic prophase, when each homolog has two sister chromatids connected by cohesins (red spheres).DSBs can be repaired through several pathways. Nonhomologous end joining is thought to be repressed inpart by controlling the localization of the involved proteins (81). Ectopic interactions do occur but arecounteracted by the process that leads to stabilization of pairing between homologs (83). Crossover (CO)formation is essential for proper chromosome segregation at meiosis I. DSBs can be repaired asnoncrossovers (NCOs) or by recombination with the sister chromatid, with the latter occurring particularlyin cases where homologs do not share homology at the site of the DSB (82) and for heterogametic sexchromosomes. During interhomolog CO or NCO recombination, DNA sequences from the initiatingchromatid (blue) are replaced locally (a few bp to several hundred bp) by sequences from the homolog( purple), resulting in a gene conversion event.

the first meiotic prophase, whereby homologsfind each other and establish connections in or-der to orient properly at the metaphase of thefirst meiotic division.

In most species, these connections are real-ized by reciprocal homologous recombinationevents, also called crossovers (COs), and visu-alized at the cytological level as chiasmata. OneCO per homolog pair is sufficient to hold themtogether in collaboration with the coordinatedestablishment of cohesion between sister chro-matids (Figure 1). In some species, such as inDrosophila melanogaster males, the homolog pair

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Crossover (CO):a recombination thatleads to the exchangeof genetic markersflanking the crossoverpoint

Chiasmata:the cytologicalmanifestations of acrossover

Gene conversion:recombination thatresults innonreciprocal transferof genetic informationfrom one DNA duplexto another

Noncrossover(NCO):a recombination eventthat is detected as agene conversion eventwithout the exchangeof flanking markers

Double-strand break(DSB): a DNAmolecule with bothstrands broken at thesame position or atclosely spacedpositions

Double-strand breakrepair (DSBR): theprocess of repairingDSBs. In meiosis,DSBs are repaired byhomologousrecombination

is held together by a distinct mechanism thatdoes not involve recombination (148). The me-chanical role of homologous recombination inchromosome segregation and the rule of theobligatory crossover imply a tight regulationof CO frequency and localization. Recombina-tion defects, i.e., altered frequencies and path-ways, can lead to abnormal chromosome seg-regation or to genomic rearrangements. This,in turn, can result in aneuploidy or geneticdefects among progeny, with consequent in-fertility (162), and genetic diseases (134, 203,216). The frequency of meiotic homologousrecombination is several orders of magnitudegreater than that of mitotic recombination [arelatively rare event often associated with oc-currence of DNA lesions or DNA replicationerrors of various origins (3)] and has a ma-jor effect on genome diversity. Meiotic recom-bination has multiple influences on genomeevolution. In particular, meiotic recombinationbreaks down combinations of alleles, allowinga more efficient elimination of deleterious mu-tations, and generates new combinations of al-leles, thus increasing the efficiency of selection(50, 240). As detailed below, CO is also associ-ated with a localized, nonreciprocal exchange ofinformation, called gene conversion; however,a fraction of meiotic recombination events aregene conversions without CO, also called non-crossovers (NCOs) (Figure 1). Gene conver-sion also has consequences for genome diversitythat are distinct from those resulting from COs(43, 240). At the molecular level, recombina-tion interactions (both for COs and NCOs) be-tween homologs contribute to homolog pairingto various extents in different species and thus toproper chromosome segregation. Remarkably,meiotic recombination occurs through an evo-lutionarily conserved program of induction andrepair of DNA double-strand breaks (DSBs).Several key steps and genes involved in DSBformation and repair are conserved, althoughdifferent mechanisms and strategies for regu-lation are observed when comparing differentspecies.

This review focuses on the formation of mei-otic DSBs and covers the various players in dif-

ferent model organisms, taking into account themost recent findings since the publications ofrecent reviews on this topic (67, 114, 178). Thestep of DSB formation is known as the initia-tion step of meiotic recombination (I keep thisterminology, although this is an empirical andsomewhat arbitrary definition), and it definesthe first detected chemical modification at theDNA level.

The review first summarizes the molecularmechanism of meiotic recombination to high-light the major steps of DSB formation andrepair and the most important features of theirregulation (see section From Formation toRepair of DNA Double-Strand Breaks: Outlineand Main Players, below). However, I do notdiscuss in detail the features of DSB repair andCO control (see 255 for review). I then presentthe current knowledge on the trans-actingfactors involved in DSB formation, thoseessential as well as those required for full DSBactivity (see sections Essential Proteins for theFormation of Mitotic DNA Double-StrandBreaks, and Control and Regulation of DNADouble-Strand Breaks, below), and on the ge-nomic sites where meiotic recombination takesplace (see sections Where Are Double-StrandBreaks Formed?, Meiotic Recombinationat Specific Genomic Regions, and HotspotDynamics and Evolution, below). This allowsoutlining of the main conserved molecularfeatures as well as the species-specific steps ofthis mechanism and future research directions.

FROM FORMATION TO REPAIROF DNA DOUBLE-STRANDBREAKS: OUTLINEAND MAIN PLAYERS

Genetic analyses in fungi led to predictionsof the molecular mechanism of meiotic re-combination, with various hypotheses for howthis event could be initiated, which interme-diates were involved, and how they could beprocessed. The double-strand break repair(DSBR) model was established on the basisof several characteristics of gene conversionand COs in fungi and on the analysis of

www.annualreviews.org • Initiation of Meiotic Recombination 565

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Topoisomerases:enzymes that alterDNA topology by areaction involvingDNA breakage andligation

DSB repair events in mitotic cells (224). Theuneven frequency of gene conversion alongchromosomes and evidence for gradients ofgene conversion, two key features of meioticrecombination, suggested the existence ofspecific sites that promote initiation. It wasproposed that observed cases of disparity ingene conversion, where genetic information istransferred preferentially from one homolog tothe other, could be explained if recombinationwas initiated by a DSB that was then processedinto a double-strand gap, making the initiatingchromatid the recipient of genetic information.The direct detection of meiotic DSBs in Saccha-romyces cerevisiae provided the first molecularevidence supporting the DSBR model (39, 173,230). Further analysis of these intermediatesled to a slight variation of the model in which

DSB is not enlarged into a gap but where onlyone DNA strand on each side of the DSBis removed (223). DSBs can be repaired byseveral pathways, the usage of which can differbetween different organisms (Figure 1).

ESSENTIAL PROTEINS FOR THEFORMATION OF MEIOTIC DNADOUBLE-STRAND BREAKS

Spo11: The Catalytic Activity forDouble-Strand Break Formation

Meiotic DSBs are catalyzed by the evolution-arily conserved Spo11 protein, which is ho-mologous to TopoVIA, the catalytic subunitof a type II DNA topoisomerase (17, 115)(Figure 2). Spo11 is composed of two domains:

Strand invasion

MRN, CtiP, ExoISpo11

removaland end

processing

Loading of Rpa, followedby Rad51/Dmc1

Spo11 and others…

DSBformation

Detection

ChIP with cross-link (before

DSB formation).

Unprocessed, highly transient

DSB fragments with Spo11

covalently bound, which can

be detected only in some MRN

(Mre11, Rad50, Nbs1) or CtiP

mutants: ChIP of Spo11,

without cross-link.

Spo11-oligo covalent complex

(wild-type meiosis), recovered

by Spo11 IP.

Processed DSB fragments with

3’ single-strand tails,

accumulate in mutants

defective for strand invasion.

Detection by ChIP of Rpa,

Dmc1, or Rad51.

Figure 2Double-strand break (DSB) formation and detection. Spo11 binds to DNA as a dimer and forms DSBs in aprocess that requires several additional proteins, generating a covalently linked Spo11-DNA intermediate.Spo11 is released as a Spo11-oligonucleotide covalent complex by endonucleolytic cleavage. 5′ ends arefurther processed, leading to long 3′ single-strand tails that are bound by Rpa and then displaced by thestrand exchange proteins Rad51 and Dmc1. These intermediates can be detected in wild-type or mutantcells, using several enrichment strategies.

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Chromatinimmunoprecipitation(ChIP): a method toidentify the DNAsequences to which agiven protein binds(directly or indirectly).The protein of interestis immunoprecipitatedfrom purifiedchromatin andassociated DNA isrecovered andanalyzed

Pseudoautosomalregion (PAR): regionon a sex chromosomethat is homologousbetween the X and theY chromosomes

(a) a DNA-binding core that contains a winged-helix domain (WHD), often referred to as acatabolite activator protein (CAP) domain be-cause it resembles the WHD domain of a CAP,and (b) a TOPRIM domain found in a variety oftopoisomerases and primases (7). The structureof archaeal TopoVIA has been solved (165) andshows that TopoVIA has a dimeric organizationin which a DSB is generated by the coordinatedaction of two TopoVIA subunits.

Similar to TopoVIA, Spo11 forms a tran-sient intermediate in which the 5′ ends of DNAare covalently linked to Spo11 through a ty-rosine that is conserved among TopoVIA andSpo11 orthologs (144). This critical tyrosinehas been identified and validated by mutage-nesis in S. cerevisiae (17), in Schizosaccharomycespombe (41), in Arabidopsis thaliana (91) and inMus musculus (24, 40). TopoVIA induces DSBswith a 2-bp 5′ overhang (37), like Spo11 (133).However, unlike the transient breaks formedin a TopoII reaction, Spo11-induced DSBsare not re-ligated, and thus DSB formation bySpo11 appears to be irreversible. Nevertheless,in some situations, Spo11 cleavage might bereversible. Indeed, chromatin immunoprecip-itation (ChIP) analysis of Spo11-DNA com-plexes in S. cerevisiae has led to the detectionof intermediates compatible with a reversiblereaction (193). The removal of Spo11 by thecombined action of several proteins, such asthe MRX complex (Mre11, Rad50, and Xrs2)in S. cerevisiae, the MRN complex (MRE11,RAD50, and NBS1) in other species, and Sae2in S. cerevisiae (named CTIP in other species),drives the irreversibility of the reaction. Thisis achieved by endonucleolytic cleavage of thestrand bound by Spo11, which leads to the for-mation of Spo11-oligos, as shown in S. cere-visiae, M. musculus (79, 164), and S. pombe (199).The outcome is a DSB end with short 3′ single-strand overhangs, which are then extended byexonucleolytic resection of the strand ending 5′

at the break (Figure 2).In mice and humans, two major SPO11 iso-

forms are detected; these differ by the presence(SPO11β) or absence (SPO11α) of 34 aminoacids in the N-terminal region. These two iso-

forms may have distinct functions, as suggestedby the observation that male mice expressingonly SPO11β are not fully fertile. This phe-notype correlates with a defect of DSB forma-tion in the pseudoautosomal region (PAR) ofthe sex chromosomes, whereas autosomal DSBformation appears mostly normal. It was thusproposed that SPO11α is required for promot-ing late DSBs, such as those normally occur-ring in the PAR (110). In A. thaliana, threeSpo11 paralogs have been identified, two ofwhich (SPO11-1 and SPO11-2) are required forDSB formation (86, 91, 215), whereas SPO11-3 is not (89, 221, 254). It is tempting to specu-late that SPO11-1 and SPO11-2 promote DSBformation as heterodimers. Several Spo11 par-alogs have also been identified in Oryza sativa(5, 102, 210). Two of these, OsSPO11-1 (257)and OsSPO11-4 (5), have been shown to be re-quired for meiosis.

Mapping Spo11-induced DSBs at nu-cleotide resolution (see sidebar, Methods forDirect Detection of Double-Strand Breaks, forDSB mapping methods) indicated that S. cere-visiae Spo11 has, at most, a low level of sequencespecificity (58, 62, 133, 180, 248, 249). Thegenome-wide map of DSB sites in S. cerevisiae,obtained by sequencing Spo11-linked oligos,showed only a slight preference for occurringin AT-rich regions (64.6% versus 60.3% inthe genome as a whole) and also a slight pref-erence for 5′-C[A/C/T] and TA at the scis-sile phosphate (180). The mapping and analysisof the characteristics of DSB sites have beenperformed in several species and are discussedbelow (see section Where Are Double-StrandBreaks Formed?, below).

Few in vitro assays have been developed toassess Spo11 function, in part because of theinsolubility of recombinant Spo11. Recently,the DNA-binding activity of an A. thalianaortholog (SPO11-1) was reported, but no DNAcleavage could be detected (208). OsSPO11-4,which is one of the five rice Spo11 orthologsand which is required for meiosis, interactswith the O. sativa TopoVIB subunit (the secondsubunit of TopoVI) in yeast two-hybrid assaysand in glutathione S-transferase pull-down


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METHODS FOR DIRECT DETECTION OFDOUBLE-STRAND BREAKS

The challenge of the molecular detection of double-strand breaks(DSBs) is twofold: DSBs are transient, and at a given location inthe genome they take place only in a small fraction of meiotic cells.DSBs can be detected by Southern blotting with various levelsof resolution (from pulsed-field gel electrophoresis to nucleotideresolution) with a sensitivity of 0.1% of total DNA molecules (36).DSBs can also be detected by end labeling and radio-labeled orfluorescent nucleotides, or by terminal transferase followed bypolymerase chain reaction amplification. These approaches havebeen used in yeast (225, 248), mice (195, 211, 258), Caenorhabditiselegans (152), and maize (185).

DSBs can be detected by ChIP of Spo11 or other recom-bination proteins (Rpa, Dmc1, Rad51). As Spo11 is transientlycovalently bound to DNA, it can be detected by ChIP withoutcross-linking.

Enrichment of DSB fragments can be achieved with the useof mutants that prevent DSB repair (i.e. sae2Δ, rad50S, dmc1Δ)and/or by purification on BND cellulose (23, 36).

DSBs can also be detected by sequencing Spo11-linkedoligonucleotides, which is the most sensitive, quantitative, andresolutive method currently available. Developed in S. cerevisiae(163, 180), this method can potentially be applied to other species.

experiments. Unexpectedly, ectopically ex-pressed OsSPO11-4 can linearize circularDNA efficiently in vitro, both in the presenceand in the absence of TopoVIB (5). Analysisof OsSPO11-4 carrying a mutation in the pre-dicted catalytic tyrosine is needed to validatethese results.

Spo11 appears to have evolved as the resultof an ancient duplication in eukaryotes of anancestral TopoVIA gene common to eukary-otes and archaebacteria. Strikingly, no Spo11has been identified in Dictyostelium discoideum,where it appears to have been lost, although agap in the genome sequence cannot be com-pletely excluded (144). Finally, it should benoted that a Spo11-independent pathway forthe initiation of meiotic recombination that en-sures chromosome segregation was detected inS. pombe strains with a mutation in the Rad2 flap

endonuclease. This endonuclease is requiredfor processing Okazaki fragments, and its ab-sence may lead to increased levels of single-strand nicks. These lesions, whether convertedinto DSBs or not, could allow initiation of mei-otic recombination events (71).

Spo11-Associated Proteins

TopoVI contains a second subunit, TopoVIB,that is required for DNA cleavage (37). A ma-jor question regarding Spo11 catalytic activ-ity is whether a TopoVIB-like subunit is in-volved. One argument in favor of such a subunitpoints out the conservation of some residuesin the Spo11 N-terminal region, which, inthe archaeal orthologs, are at the interfacebetween TopoVIA and TopoVIB (52, 85).Orthology searches have not identified anySpo11-interacting subunit so far. However, aTopoVIB subunit was identified in plants, butit is not required for meiotic recombination (89,90, 221, 254).

Several proteins that interact directly orindirectly with Spo11 and are required formeiotic DSB formation (as indicated by theabsence of DSBs in the corresponding mu-tants) have been identified in S. cerevisiae (114)(Table 1, Genes essential for DSB). Only someof these Spo11-associated proteins are con-served. Various approaches, either biochemi-cal to detect protein interactions or cytologicalto monitor nuclear localization, have been de-veloped to analyze their properties. At the be-ginning of meiotic prophase, a proteinaceaousstructure assembles to build chromosome axesfrom which emanate chromatin loops. As pre-sented in the section Control and Regulationof DNA Double-Strand Break Formation, thisorganization plays an important role for DSBformation and repair. Thus, nuclear localiza-tion of several proteins and their dynamicsduring meiotic prophase has provided key in-formation regarding their roles in meioticrecombination. On the basis of these various ap-proaches performed in S. cerevisiae and S. pombe,the Spo11-associated proteins can be organizedin three different subcomplexes (Figure 3).

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Table 1 Genetic control of double-strand break (DSB) formationa

Saccharomycescerevisiae

Schizo-saccharomyces

pombeArabidopsis

thalianaCaenorhabditis

elegansDrosophila

melanogasterMus

musculus ActivityDSBnumber

140–170 indmc1Δ, 160in wild type

150–294 inwild type, upto 342 inmdn1−/−, and381 in atr−/−

12–46 inrad-54

14 in wildtype, up to24 in spn-A(rad51),spn-B (rad51paralog), andspn-D (rad51paralog)

230–400 inmales,250–370 infemales

Method DSBfragmentsor Spo11-oligos

DMC1 orRAD51 foci

RAD51 fociand Tunelassay

γH2AV foci RAD51 andDMC1 foci

Reference 36 202, 42, 232,125

152, 171, 198 150, 109 Seereferencesin 13

Genesessentialfor DSB

SPO11 rec12 SPO11-1 spo-11 mei-w68 Spo11 Transesterase

SPO11-2 Transesterase

(SPO11-3) Transesterase

rec6

SKI8 rec14 (SKI8) Wdr61, nt

REC102

REC104

REC114 rec7 (PHS1) Rec114, nt

MEI4 rec24 PRD2 Mei4

MER2 rec15

CDC7 hsk1 AT4G16970,nt

cdc-7, nt l(1)GO148, nt Cdc7, nt Proteinserine/threoninekinase

DBF4 dfp1, nt chiffon, nt DbF4, nt

DFO

mde2

RAD50 (rad50) Rad50, nt DNA binding,ATP binding

MRE11 (mre11) (Mre11) Endonuclease,exonuclease

XRS2 (nbs1) (Nbs1)

PRD1 Mei1

PRD3

mei-P22

trem(Continued )


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Table 1 (Continued )

Saccharomycescerevisiae

Schizo-saccharomyces

pombeArabidopsis

thalianaCaenorhabditis

elegansDrosophila

melanogasterMus

musculus ActivityGenesneededfor wild-typeDSBactivity

HOP1 hop1 (ASY1) htp-3 Hormad1

MEK1 mek1 Kinase

rec25

rec27

(Rad21L) Kleysin

REC8 rec8 REC8/Syn1,nt

rec8, nt (Rec8) Kleysin

SCC3 rec11 SCC3, nt (scc3) sa, nt Stag3, nt Cohesincomplexsubunit

TEL1 tel1, nt (ATM) atm-1, nt atm Atm PI3 kinase

MEC1 rad3, nt (ATR) atl-1, nt (atr) Atr, nt PI3 kinase

SET1 set1 ATX1,ATX2, nt

set1/mll, nt Set1A, nt Methyl-transferase

Set1B, nt Methyl-transferase

Prdm9 Methyl-transferase,DNA binding

SPP1 spp1, nt Cfp1, nt cfp-1, nt cfp1, nt Cfp1, nt PHD domain,DNA binding

PCH2 AT4G24710,nt

pch-2, nt pch2, nt (Trip13) AAA+ ATPase

aThe estimated number of meiotic DSBs in various species with the method of detection is shown. Genes involved are displayed in two parts: thoseessential for DSB formation and those needed for wild-type DSB activity. Genes in parentheses are orthologs or paralogs not required for DSB formation.Abbreviation: nt, role in meiotic DSB formation not tested.

The first complex is composed of Spo11 andSki8, a WD (tryptophan–aspartic acid) repeatprotein that directly interacts with residues inthe C-terminal domain of Spo11, as indicatedby yeast two-hybrid assays (8). The interac-tion between the S. pombe orthologs of Spo11(Rec12) and Ski8 (Rec14) was also detected bytwo-hybrid assay (217) and coimmunoprecipi-tation (CoIP) (155). Ski8 is evolutionarily con-served, expressed in meiotic and nonmeioticcells, and involved in RNA processing in S.cerevisiae mitotic cells (6). The requirement forSki8 in DSB formation has been shown in S.cerevisiae, S. pombe (70), and Sordaria macrospora(226), but its precise role is unknown. Ski8 is

not required for meiotic DSB formation in A.thaliana (108), raising the question of its func-tional conservation for meiosis outside fungi.WDR61, the mammalian Ski8 ortholog, is partof the PAF complex (involved in transcriptioninitiation and elongation) and of the SKI com-plex (RNA surveillance and processing) (263).Whether WDR61 also plays a role in meioticDSB formation is unknown.

The second complex is composed of Rec102and Rec104 in S. cerevisiae (107, 112, 113, 201),but orthologs have not been identified in otherspecies. These two proteins interact with eachother, and Rec104 also interacts with Spo11.Rec102 and Rec104 bind to chromatin and are

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A. thaliana

Prd2

DFO

Spo11-2Spo11-1

Prd1Prd3

M. musculus

Mei4

Mei1

Spo11Rec114

S. pombe

Rec14

Rec6

Mde2

Rec7

Rec24

Rec15

Rec12

S. cerevisiae

Rec114

Mei4

Xrs2Rad50

Mre11

Rec104

Rec102

Ski8

Spo11

Mer2

Figure 3Proteins that are essential for double-strand break (DSB) formation and their interactions in Saccharomyces cerevisiae, Schizosaccharomycespombe, Arabidopsis thaliana, and Mus musculus. Interactions were detected by coimmunoprecipitation (red arrows) and in yeast two-hybridassays ( gray or black arrows). The two-hybrid-based interaction map for S. cerevisiae proteins is from Maleki et al. (142), with gray arrowsindicating interactions detected in mitotic cells and black arrows indicating interactions detected only in meiotic cells.

required for Spo11 dimerization, DNA bind-ing, and efficient nuclear retention (113, 193,205). Rec102 and Rec104 bind to chromatinalong chromosomes, with some preference forthe chromosome axis; however, they are notpreferentially enriched near DSB sites and theirrole remains to be determined (113, 182). InS. pombe, Rec6 has been identified as an ad-ditional partner that is essential for meioticDSB formation; it forms a complex with twoother proteins, Rec12 and Rec14 (61). Rec6does not interact with Rec12 or Rec14 indi-vidually in yeast two-hybrid assays but does sowhen they are coexpressed (155). Yeast two-hybrid assays and CoIP experiments suggest

that the S. cerevisiae Rec102-Rec104 complexmight form a bridge between Spo11 and athird complex (Rec114-Mei4-Mer2) (8). Sim-ilarly, the S. pombe Rec12-Rec14-Rec6 com-plex interacts with the S. pombe orthologs ofRec114-Mei4-Mer2, which are named Rec7-Rec24-Rec15 (155) (see below).

The Rec114-Mei4-Mer2 complex is re-quired for DSB formation (129, 142, 205), andMer2 colocalizes with Rec114 and Mei4 atchromosomal axis sites (129, 142). Mer2 is a keycontrol point in the function of this complexand provides a link between DNA replicationand DSB formation. DSB formation occursafter S phase, and although replication is


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Synaptonemalcomplex: the proteinstructure that stablypairs homologouschromosome axes fromend to end duringmeiotic prophase via aprocess called synapsis

γH2AX: thephosphorylated formof the histone H2Avariant H2AX onserine 139; detectedupon DSB formationafter activation of theATM/ATR kinases

not essential for DSB formation (97), DNAreplication and DSB formation are temporallycoupled (28). Mer2 is phosphorylated by twokinases that are required for DNA replication:Cdc28 (in association with the B-type cyclinsCbl5 and Clb6) (94) and Cdc7 (in interactionwith Dbf4) (204, 237). Phosphorylation hasbeen detected at several sites, two targetedby Cdc28 and several others by Cdc7. Someof the residues phosphorylated by Cdc7 areprimed by Cdc28-dependent phosphorylationat residue S30 (157). Mer2 phosphorylationis required for Mer2 interaction with Rec114(94) and the recruitment of Rec114, Mei4,and Spo11 to DSB sites (204). Importantly,in a clb5-clb6 double mutant or in a nonphos-phorylatable mer2S30A mutant, Mer2 bindingto axis sites still occurs efficiently, whereasbinding of Rec114 and Mei4 is mostly lost(182). Mer2 regulation is thus thought toprovide a link between DNA replication andDSB formation, suggesting that other proteinsof the Spo11-associated complexes are loadedonto chromosome axes during meiotic S phase(28, 157). Mer2 association with axis sitesdoes not require Mei4 and Rec114, which isconsistent with the idea that these proteins arerecruited by Mer2 (182). Rec114-Mei4-Mer2association with the chromosome axis istransient and declines after DSB formation(182).

In S. pombe, Hsk1, the ortholog of Cdc7,may play a similar role because it is essentialfor DSB formation (174) and is required forSpo11 recruitment to DSB sites (204). The in-teraction between S. pombe Rec24 (Mei4) andRec7 (Rec114) is conserved and is also neededfor DSB formation (25, 217). Recent assayshave shown that Rec24, Rec7, and Rec15 (apossible Mer2 ortholog) also form a complexin which Rec7 directly interacts with Rec24.Rec15, Rec7, and possibly Rec24 are detectedboth on axis and DSB sites. Rec15 recruit-ment to DSB sites depends on Rec24 and Rec7but not on Rec12, suggesting that the Rec7-Rec24-Rec15 complex may recognize somespecific features of DSB sites. In addition, the

Mde2 protein, which lacks an S. cerevisiae or-tholog, is essential for DSB formation, inter-acts with Rec15 to stabilize the Rec7-Rec24-Rec15 complex, and, interestingly, also bindsto Rec14, thus bridging the two subcomplexes(155).

Mei4 and Rec114 are conserved in mosteukaryotes (124). Mouse MEI4 is requiredfor meiotic DSB formation and interacts withREC114 (124). MEI4 is localized in multipleand discrete foci on the axes of meiotic chro-mosomes, independent of SPO11 (124). TheA. thaliana Mei4 ortholog PRD2 also is re-quired for DSB formation (59). However, Zeamays and A. thaliana Rec114 orthologs (Phs1)are not required for DSB formation but are re-quired for DSB repair and homologous pair-ing (185, 197). These results suggest that inplants, Rec114 has functions distinct from thosein fungi and mammals. In this regard, it isintriguing to note that Mei4 and Rec114 or-thologs have not been found in S. macrospora,D. melanogaster, and Caenorhabditis elegans,three species that lack Dmc1, Hop2, and Mnd1,which are meiosis-specific proteins involvedin strand exchange in both yeast and mam-mals (143). It is tempting to speculate thatMei4 and Rec114 may play a role in coordi-nating DSB formation with later steps in DSBrepair.

A few additional factors have been identifiedby using genetic approaches. M. musculus Mei1and its A. thaliana ortholog PRD1, which arerequired for DSB formation (60, 130), encodefor proteins containing Armadillo repeats withsimilarities to importins, but their activitiesare unknown. In plants, A. thaliana PRD3(59) and its O. sativa ortholog PAIR1 (170)are required for DSB formation, as is theproduct of the A. thaliana DFO gene (260). InD. melanogaster, MEI-P22 is required for DSBformation and localizes to discrete foci atmeiotic chromosomes after the formation ofthe synaptonemal complex but before the ap-pearance of the phosphorylated form of histoneH2A variant H2AV (γHis2AV, the equivalentof mammalian γH2AX), which forms in

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Lateral element:the proteinaceousstructure that definesthe chromosome axisat the beginning ofmeiotic prophase

Cohesin: a proteincomplex that holdssister chromatidstogether

response to DSB formation (132, 150). Forma-tion of MEI-P22 foci requires Trade Embargo,a chromatin-associated protein with C2H2 zincfingers that is also needed for DSB formation(126).

It should be noted that in many organisms,DSB formation deficiencies are not detectedby direct monitoring of DSB molecules butby cytological detection of γH2AX and/oraccumulation of DSB repair proteins, such asRAD51 or DMC1, that bind to the single-stranded DNA present at DSB ends. Theseassays have limited sensitivity, and they cannotdistinguish defects in DSB formation fromdefects in the early stages of DSB processing.These limitations should be taken into accountwhen interpreting data. In this context, one re-liable assay that has been often used to identifymutations that lead to a loss of DSB formationinvolves the suppression of the meiotic chro-mosome fragmentation seen in DSB repairmutants.

CONTROL AND REGULATIONOF DNA DOUBLE-STRANDBREAK FORMATION

Role of the Chromosome Structureand Organization

Many observations have provided insightssuggesting that the chromosome organizationplays an important role in the regulation of var-ious features of meiotic recombination. DSBrepair is regulated in such a way to control thechoice of the template (the sister chromatid, orthe homolog) for DSB repair and to regulatethe number of COs. Meiotic chromosomes areorganized in loops anchored to the axis (calledthe lateral element at the beginning of meioticprophase), and DSB repair takes places in thecontext of the axis (264, 265). However, studiesin S. cerevisiae indicate that DSBs mostly occurat sites not constitutively axis-associated butlocated within loops emanating from the axis,suggesting that these regions are recruited tothe axis before or at the time of DSB formation

(21, 119). As several DSB proteins (particularlythe Rec114-Mei4-Mer2 complex) are locatedon the axis, this may suggest that DSBs occuronce the DSB sites are tethered to the axis.The next paragraphs discuss the role of axis-associated proteins in DSB formation and pos-sible ways they might interact with DSB sites(27).

Chromosome axis: HORMA domain–containing proteins. S. cerevisiae Hop1 is ameiosis-specific protein that is an importantfactor in DSB formation and also promotesinterhomolog rather than intersister recombi-nation during DSB repair. Hop1 contains aHORMA (Hop1p, Rev7p, and MAd2) domain,which has been suggested to recognize chro-matin states that result from DNA adducts,DSBs, or detachment from the spindle, and thatis thought to act as an adaptor to recruit otherproteins. Hop1 is localized along meiotic chro-mosomes at the beginning of meiotic prophase,before and independent of DSB formation,in complex with a second axis-associated pro-tein, Red1. In hop1 mutants, DSBs are re-duced to 5% to 10% of wild-type levels (146,245). Chromatin immunoprecipitation (ChIP)analysis reveals a nonrandom Hop1 associationwith chromosomes, with Hop1-enriched do-mains corresponding to high-frequency DSBdomains (182). The Spo11 accessory pro-teins Rec114/Mei4/Mer2 are also enriched atthese domains, and Mer2 is not recruitedto chromosomes in the absence of Hop1(182).

In addition to its role in DSB formation,the Hop1-Red1 complex is also required foractivation of the Mek1 kinase, which repressesrecombination between sister chromatids(167). The Hop1-Red1, and thus the Mek1,complex is thought to regulate the choicebetween interhomolog and intersister recom-bination, in part by counteracting the sisterchromatid recombination-promoting activityof the cohesin Rec8 and by inhibiting thestrand transfer activity associated with theRad51 protein (38, 117, 168). Mutants lacking


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Rec8, a meiosis-specific subunit of cohesin,show altered DSB formation, with differentregions showing decreased, unchanged, orincreased DSB levels (21, 117, 123). Inter-estingly, Hop1 localization is dependent onRec8 in some regions but not others, andthose regions where Hop1 is retained in a rec8mutant strain also retain Rec114 associationand DSB formation. This suggests that Rec8helps to recruit, stabilize, and/or constrainHop1 at some regions but not at other regionsin which Hop1 can be loaded efficiently in theabsence of Rec8 (182).

Some of these features seem to be conservedin other species. In S. pombe, mutants lackingthe Rec8 or Rec11 cohesin components showregion-specific reductions in meiotic recombi-nation, and DSBs are not detected in mutantsthat lack Rec10, a putative ortholog of S. cere-visiae Red1 (69). S. pombe Rec10 and Hop1 colo-calize on chromosome axes that are called linearelements, and their localization is altered in theabsence of Rec8 (136). Cytological studies in-dicate that Rec10, but not Hop1 or Mek1, isrequired for Rec7 (Rec114) localization to theaxis (135). This effect is likely to be mediatedthrough Rec15, as Rec15 is required for Rec7localization to DSB sites, and Rec10 and Rec15have been shown to interact in a yeast two-hybrid assay (155). DSB formation is partiallyreduced in hop1 or mek1 mutants (53% and 70%of the wild-type DSB levels, respectively) andis further reduced in the double mutant (22%)(128, 199).

In M. musculus, two Hop1 orthologs havebeen identified: HORMAD1 and HORMAD2(77, 181, 243). HORMAD1 localizes to unsy-napsed chromosome axes at leptotene and is dis-placed upon synapsis formation. In Hormad1−/−

mutants, DSB levels are reduced to 25% of thewild-type level, and the checkpoint response,which in female meiosis leads to the elimina-tion of defective oocytes, is strongly reduced.These phenotypes place Hormad1 at the inter-face between DSB formation, DSB repair, andthe checkpoint response (55, 243). The role ofHormad1 may indeed be similar to that of Hop1in S. cerevisiae, as it is required for the stabi-

lization of MEI4 foci on the chromosome axesin mouse spermatocytes (R. Kumar, K. Daniel,A. Toth, B. de Massy, unpublished data).HORMAD2 is a HORMAD1 paralog with avery similar cytological localization with slightdifferences, such as its specific accumulation onthe sex chromosomes at diplotene, that sug-gest roles distinct from those of HORMAD1.Indeed, Hormad2−/− mutant female mice arefertile, and males do not show defects in DSBformation and repair but are deficient in ATRrecruitment to and silencing of the unsynapsedregions of sex chromosomes (242).

In C. elegans, several HORMAD-like pro-teins have been identified, and all are struc-tural components of the meiotic chromosomeaxis (259). In particular, HTP-3 is associ-ated with chromosome axes throughout meioticprophase (141) and is required for DSB forma-tion (84). HTP-3 is also involved in cohesinloading (206). Interestingly, HTP-3 interactswith MRE11, thus providing a potential link be-tween DSB formation and repair. HTP-3 alsoforms a complex with HIM-3, another Hop1ortholog that is involved in pairing and synapsis,and potentially regulating the use of the sisterchromatids for DSB repair (53). In C. elegans, aconnection between cohesins and HORMAD-like proteins is suggested by the mislocaliza-tion of HTP-3 in scc3 mutants also deficient forREC8 loading on chromosomes (184, 238). Alarge number of RAD51 foci are detected in scc3mutants, indicating that HTP-3 axis associationis not required for DSB formation (84).

The ASY1 protein of A. thaliana has limitedhomology with S. cerevisiae Hop1, and DSBformation appears to be unaltered in asy1 mu-tants. However, the ASY1 protein may retainHop1 function in regulating partner choicein recombination (202). The O. sativa ASY1ortholog (PAIR2) has a localization patternsimilar to that of HORMAD1 and, like ASY1,is essential for homolog synapsis, but itsinvolvement in DSB formation has not beeninvestigated (169). However, ASY3, a proteinthat interacts with ASY1, is required for fullDSB levels (73). A functional interaction withcohesins is also suggested because SWI1, a

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protein required for cohesion between sisterchromatids during meiosis, is also requiredfor DSB formation and for normal ASY1localization (151).

The functional conservation betweenmouse and C. elegans HORMAD proteins andS. cerevisiae Hop1 is further documented bystudies of the role of TRIP3 and Pch2, twoAAA+ ATPase orthologs that are involved inmaintaining the normal distribution of HOR-MAD/Hop1 proteins on the chromosomeaxis and that are required for the pachytenecheckpoint that is triggered by failures inhomolog synapsis (18, 32, 243). S. cerevisiaepch2 mutants are partially defective in DSB for-mation, with different defects in mutants withdifferent DSB end-processing defects, thussuggesting a regulatory role in DSB formationthat involves a feedback from DSB processing

signals (72). This issue of feedback control inDSB formation is further discussed below (seesection Coordination Between Formation andRepair of DNA Double-Strand Breaks).

Linking DNA double-strand break sitesto chromosome axes: the distinct strate-gies used in Saccharomyces cerevisiae andin Schizosaccharomyces pombe. The axis local-ization of the Rec114, Mei4, and Mer2 proteins,which are required for Spo11 activity, raisesthe question of how DSB sites and Spo11 arebrought to the axis (Figure 4). In S. cerevisiae,this is mediated by the Spp1 protein, a memberof the COMPASS chromatin-modifying com-plex that interacts preferentially with promoterregions in mitotic cells (207, 2, 213). Spp1 doesthis by interacting both with the axis-associatedprotein Mer2 (see above) and, through its plant

Hop1/Rec10

Axis Axis Axis

Rec25,Rec27,Mug20

S. pombe

Rec15, Rec7,Rec24, Mde2Rec8/

Rec11

?

Spo11

NDRTF?

Spp1Hop1/

Red1Mer2

Mei4

Rec114

Rec8

Chromatinloops

S. cerevisiae

Spo11

NDR

TSS

Set1 complex

Pol II

?

MEI4

M. musculus

Hormad1

SPO11

PRDM9

ZnF

SET

Figure 4Guiding Spo11 and connecting double-strand break (DSB) sites to an axis. In Saccharomyces cerevisiae, Spo11 binds tonucleosome-depleted regions (NDRs) adjacent to transcription start sites (TSSs). DSB sites interact with the axis through Spp1. InSchizosaccharomyces pombe, Spo11 binds preferentially to NDRs, possibly with the contribution of transcription factors (TFs), at least atsome sites. Several proteins essential for DSB formation are associated with the chromosome axis and have the ability to interact withDSB sites. This interaction may take place well in advance of DSB formation or at the same time. In Mus musculus, PRDM9 binds tospecific sites in the genome through its zinc finger (ZnF) domain, and is hypothesized to recruit SPO11. These regions are predicted tobe tethered to an axis where MEI4 is localized but are done so through an unknown mechanism. H3K4me3 deposited either by Set1 inS. cerevisiae or by PRDM9 in M. musculus is depicted as a magenta star.


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Histone H3trimethylated lysine4 (H3K4me3): thismodification is knownto be enriched inaccessible chromatin,often neartranscription start sitesand some transcriptionenhancers

Nucleosome-depleted regions(NDRs): regions ofthe genome with lownucleosome density asindicated byhypersensitivity todigestion by nucleases

Condensin: a proteincomplex that playsroles in chromosomeorganization andcompaction

homeodomain (PHD) finger, with histone H3trimethylated lysine 4 (H3K4me3), a chromatinmark often found near nucleosome-depletedregions (NDRs) in chromatin that are bound bySpo11 (27). Spp1 is required for a normal levelof DSB formation, but DSBs are still detectedin an spp1� strain. In spp1� strains, DSBs occurat very low levels at sites used in wild-type yeastcells, but strong DSB sites that are still Mer2-dependent occur at new positions. These newlocations have been proposed to be chromatinloop regions with a propensity to interact withthe chromosome axis.

In S. pombe, the association between DSBsites and chromosome axes has been recentlydescribed by two studies that highlight severalkey components (76, 155). Rec25 and Rec27(147), two linear element components withno ortholog identified in S. cerevisiae, are re-quired for full levels of DSB formation (56).On the basis of cytological and mass spectrom-etry analyses, Rec10, Hop1, Rec25, and Rec27may form one or several complexes, possiblywith Mug20 (147, 214). Interestingly, genome-wide ChIP-Chip showed that Rec10 is rel-atively uniformly distributed along chromo-somes, whereas Rec25, Rec27, and Mug20 arehighly enriched near most DSB sites, even inthe absence of Rec12 (76). Rec25 and Rec27thus appear to recognize some as yet unknownfeature of DSB sites. In addition, Mde2, Rec15,and Rec24, which interact with the DSB cat-alytic machinery (see above) are also associ-ated with DSB sites in the absence of Rec12(155). Analysis of the S. pombe genome-wideDSB map (see below) suggests that H3K4me3does not play an essential role for DSB forma-tion, and that, unlike in S. cerevisiae, the DSB-axis interaction is not expected to require Spp1(Figure 4).

Thus, in both yeasts, DSB sites have two keyproperties: (a) They are favorable substrates forSpo11-Rec12 binding, in part because they arein regions of nucleosome depletion (see below);and (b) they have properties that promote theirrecruitment to the chromosome axis, althoughdifferent mechanisms for recruitment appear tobe used in S. cerevisiae compared with S. pombe.

Condensins and axis length. Condensins,another component of the chromosome struc-ture, play an important role in meiotic DSBformation in C. elegans. Several condensins arepresent in meiotic cells, and the condensin Icomplex (246) affects DSB formation. Specif-ically, its disruption leads to increased axislength that is correlated with a higher num-ber and altered localization of DSBs, whichare scored as RAD51 foci and revealed by aTUNEL assay (152). Studies in many specieshave reported correlations between axis lengthand CO activity, and this phenomenon has beendescribed in detail in humans and mice (120).It is possible that an extended axis may leadto smaller and more numerous loops, whichmay provide more potential for DSB activityand/or alter CO control. Proteins other thancondensins may also be involved in this regu-lation. For instance, a correlation between axislength and loop sizes has been shown in mice inwhich Sycp3, a component of the meiotic chro-mosome axis, and Smc1β, a meiotic-specific co-hesion subunit, were knocked out (172). Theimpact of these mutations on DSB formationwas not assessed.

Further evidence for an association betweenchromosome organization and recombinationactivity comes from analysis of the M. musculusdomesticus PAR of the sex chromosomes. Thisregion is approximately 800 kb long, whichcorresponds to about 1% of the autosomalgenome, but it is the locus of an obligate COduring male meiosis that is required for sexchromosome segregation. DSBs occur in thePAR at a rate, per kilobase of DNA, that is20-fold higher than in autosomes. Moreover,measurement of loop sizes and axis lengthshow a marked difference between the PARand autosomes: PAR loops are threefold to sev-enfold shorter than in autosomes and are thuspredicted to be more numerous, whereas theaxis length relative to DNA content is tenfoldgreater in the PAR than in autosomes (110).If the number of active DSB sites per loopis a limiting factor, this specific organizationmay allow for a higher DSB density. Similarvariation in properties of chromosome

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organization on autosomes may also explainor contribute to observed regional variationsin DSB activity, which remain largely notunderstood.

Global Effects of ChromatinModifications

In S. cerevisiae and S. pombe, several chromatinmodifiers affect local chromatin structure atDSB sites, with consequences for DSB forma-tion (see below). Some modifications may alsohave chromosome-wide functions and effects.For instance, C. elegans HIM-17 (a protein withno identified activity) is required for normalDSB formation, and him-17 mutants show re-duced or delayed DSB formation and histoneH3 lysine 9 (H3K9) dimethylation (196). In themutant, synapsis (which is DSB independent inC. elegans) is normal, but RAD51 foci and chi-asmata are not detected. The HIM-17 proteinlocalizes to chromatin in germ cells. Him-17genetically interacts with Rb, which can mod-ify chromatin through interactions with his-tone modifiers. Given that him-17 is requiredto maintain a repressive mark (H3K9me2), it isnot expected to be directly involved in SPO11accessibility. Rather, the phenotype observed inhim-17 mutants may be indirect, for example,with an inappropriate recruitment and titrationof DSB components to regions normally re-pressed and where the mutants cannot be active(196).

Similarly, on the basis of analysis of RAD51foci, the C. elegans XND-1 protein is alsorequired for DSB formation (233). In addi-tion, different xnd-1 mutant phenotypes are ob-served on different chromosomes. CO distribu-tions are altered on chromosomes I and X, butCO frequencies and RAD51 foci are reducedonly on chromosome X. The X chromosome–specific reduction of DSBs and COs is surpris-ing because in wild-type worms, the XND1protein is enriched only on autosomes. Thexnd-1 mutant also shows increased histoneH2A lysine 5 acetylation, which is catalyzedby the TIP60 histone acetylation complex,whereas other histone modifications were un-

affected. As TIP60 is recruited to DSBs dur-ing repair, some of the effects observed inxnd-1 mutants could be at the level of DSBrepair.

Coordination Between Formation andRepair of DNA Double-Strand Breaks

DSB end recognition and processing is one ofthe first steps of DSB repair after DSB for-mation. DSB ends, to which Spo11 is cova-lently attached, are recognized by the MRX(MRN) complex and processed via Spo11-oligo removal, followed by strand resection(Figure 2). The MRX (MRN) complex is re-quired for DSB formation in S. cerevisiae (4)and C. elegans (46) but not in S. pombe (256),A. thaliana (22, 194), Tetrahymena thermophila(140), Coprinopsis cinereus (1), D. melanogaster(150), or M. musculus (45).

In S. cerevisiae, Mre11 is associated with DSBsites independent of DSB formation and Rad50.This interaction requires all Spo11 accessoryproteins and involves the C-terminal end ofMre11, which contains a DNA-binding domain(26, 29). It has been suggested that Mre11 playsa role in remodeling chromatin at DSB sitesbecause mre11 mutants display reduced micro-coccal nuclease sensitivity at such sites (175).This property of the MRX complex in S. cere-visiae may contribute to the coordination be-tween DSB formation and repair.

One poorly elucidated aspect of DSBregulation involves the control of their timing.Evidence for the coordination of DNA replica-tion and DSB formation via S. cerevisiae Mer2(see above) provides insights into “turn-on”control, but little is known about how DSBsare “turned off.” A first clue as to the natureof this regulation comes from the recentdiscovery of negative feedback control on DSBformation, mediated by ataxia telangiectasiamutated (ATM) (127). ATM, which belongsto the superfamily of phosphatidylinositol3-kinase–related kinases, is recruited to DSBsand activated by the MRN complex throughdirect interaction with NBS1. ATM promotes


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a variety of events in response to DSBs, in-cluding autophosphorylation, phosphorylationof the H2AX histone variant, and activation ofMRE11 and of the CHK2 effector kinase (101,241). ATM also appears to repress DSB forma-tion in mice, as evidenced by increased levelsof SPO11-oligos in Atm−/− mutants (127).ATM-mediated negative regulation of meioticDSB formation has also been observed inD. melanogaster through the analysis of histoneH2AV phosphorylation (109). As ATM activa-tion occurs in the vicinity of DSBs, ATM maylimit the formation of DSBs at nearby sites onthe same chromatid, on the sister chromatid,or on the chromatids of the homolog. Studiesin S. cerevisiae suggest that DSB formationis constrained to occur at a given site onlyonce per pair of sister chromatids and thatconstraints also extend to the same site on thehomolog (261). The two S. cerevisiae DNAdamage-response kinases, Mec1 and Tel1(homologs of ATR and ATM, respectively),appear to be involved in this inhibition,although mutants in the two kinases havedifferent effects, with DSB levels being slightlyreduced in mec1 and unchanged in tel1 mutants(261).

WHERE ARE DOUBLE-STRANDBREAKS FORMED?

Several tools have been developed to de-tect meiotic DSBs and genome-wide high-resolution maps of DSBs are available forS. cerevisiae, S. pombe, and M. musculus (see side-bar, Methods for Direct Detection of Double-Strand Breaks). However, in several organisms,DSBs have not been directly monitored, and in-formation about initiation sites is obtained fromCO and NCO maps that cannot be directly ex-trapolated to DSB activity without additionalhypotheses. Below, I discuss data obtained us-ing these various approaches, with a specificfocus on the most recent high-resolution andgenome-wide findings that provide novel in-sights into DSB site localization.

A general observation is that recombina-tion events are not randomly distributed. Whenevents are examined at high resolution (single-nucleotide to kilobase level), hotspots andcoldspots for recombination and DSB forma-tion are observed; when events are observed atlow resolution (regions or domains of chromo-somes), regions of high and low DSBs and re-combination, called jungles and deserts, respec-tively, are observed. The presence of hotspotsand coldspots suggests highly localized proper-ties of the genome that are favorable to DSBactivity, and the presence of jungles and desertssuggests that features of chromosomal domainsmodulate DSB activity. Progress has been madein understanding hotspot localization, but thecontrol at the level of domains remains poorlyunderstood.

The term hotspot itself can be misleading, aslevels of DSBs or recombination at sites calledhotspots can vary over several orders of mag-nitude, and this calls into question the criteriaused to identify a given locus as a hotspot or toestimate the number of hotspots in the genome.Some researchers use as comparative criteriathe recombination activity of the adjacent re-gions, whereas others use the genome-wide av-erage. Because the term hotspot has been sowidely used, it is retained in the rest of thearticle, keeping in mind the above-mentionedcaveats.

Double-Strand Break Maps inSaccharomyces cerevisiae: TheOpportunistic Spo11

From the first detection of meiotic DSBs(222) to the recent high-resolution genomemaps, many laboratories have contributedto the description of the DSB landscape inS. cerevisiae by developing and optimizingseveral approaches (see sidebar, Methods forDirect Detection of Double-Strand Breaks).Sequencing Spo11-oligos led to the identifica-tion of 3,604 DSB hotspots, which are definedas having Spo11-oligo densities approximatelytwofold higher than the genome-wide average

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(180). A striking feature of these hotspots istheir strong correlation with transcriptionpromoters (88.2% overlap). DSB sites extendover small intervals (73.4% are 50–300 bp insize) and are located in NDRs, which is inagreement with the earlier evidence for theimportance of chromatin accessibility at S.cerevisiae hotspots (176, 247). Hotspots have awide range of activity with continuous varia-tion, and low levels of DSBs can be detectedin non-hotspot regions, outside transcriptionpromoters. Spo11 has no or little DNAsequence specificity (see above), and DNAaccessibility appears to be one of the majorfeatures that drives Spo11 to its substrate. Infact, DSB activity can be targeted to specificDNA sequences by expressing a Spo11 fusionprotein that contains the Gal4 DNA-bindingdomain (186). Wider hotspots tend to havelarger NDRs and also tend to have higher DSBactivity (180). Moreover, factors that modulatechromatin features and accessibility, such astranscription factors and chromatin modifiersand remodelers, have been shown to influenceDSB activity (189). One additional componentthat plays a major role, through binding toH3K4me3 and H3K4me2 and interactingwith Mer2 (see above) (Figure 4), is theprotein Spp1, which appears to tether DSBsites into the context of chromosome axes.Each parameter analyzed independently—suchas accessibility, measured by nucleosomeoccupancy or H3K4me3 enrichment—is not agood indicator of the quantitative levels of DSBactivity (31, 180, 228). This may be explainedby the combined requirement for chromatinaccessibility and H3K4me3 enrichment.Genome-wide detection of COs and NCOs bygenotyping tetrads has also provided a map ofrecombination events that is in agreement withthe high-resolution DSB maps (145).

Double-Strand Break Maps inSchizosaccharomyces pombe

Hotspots have been mapped in S. pombe byChIP of Rec12, the S. pombe Spo11 homolog

(54, 139). In one study, a rad50S mutantwas used to recover Rec12-DNA complexeswithout cross-linking, whereas in anotherstudy, performed using a wild-type strain,chromatin was cross-linked. Of note, inS. pombe, no difference in DSB levels wasdetected in rad50S and wild-type strains(100). The distance between DSB hotspots inS. pombe is greater than in S. cerevisiae (1 DSBhotspot per 65 kb on average in S. pombe, com-pared with 1 per 3.4 kb in S. cerevisiae), and thenumber of DSB hotspots is correspondinglyabout 10-fold lower. In S. pombe, DSB hotspotsare not correlated with transcription promot-ers, and instead the strongest DSB hotspotsare enriched in large intergenic regions of3 kb or more that include clusters of NDRs, anenrichment most pronounced at the strongestDSB sites. Overall, most DSBs (95%) overlapwith NDRs (57). However, as in S. cerevisiae,nucleosome depletion is not sufficient to form ahotspot because 2,973 NDRs can be defined inthe S. pombe genome. A correlation was also ob-served between the presence of a DSB hotspotand the level of noncoding RNAs transcribedfrom a locus, which may be due to the greaterabundance of noncoding RNAs transcribedfrom large intergenic regions (236). Geneticand molecular analyses of a few hotspotshave identified a meioses-specific change inchromatin structure near DSB sites and haveidentified roles for transcription factors in influ-encing DSB activity. The most detailed analysiswas developed at the ade6-M26 hotspot, whereactivity depends on the presence of a consensusto the binding site of the heterodimeric tran-scription factor Atf1.Pcr1 (220, 234). Severalchromatin modifiers and remodelers also influ-ence DSB activity (96, 250). Complementaryto these analyses, the search for new hotspotsled to the identification of hotspot-specificDNA motifs, some of which are recognizedby transcription factors (218, 219). On thebasis of these observations, it was hypothesizedthat a family or families of transcriptionfactors might actually recruit Rec12, eitherdirectly or indirectly (234, 235). No specific


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enrichment for H3K4me3 is observed at DSBsites in S. pombe, and the role of Set1, if any,in DSB formation in S. pombe remains to beunderstood (251).

Double-Strand Break Map in Musmusculus: A Molecular Strategy toTarget Double-Strand Break Activity

Genome-wide mapping of DSB hotspots in themouse was recently performed by ChIP forthe recombinases DMC1 and RAD51 in chro-matin purified from testis extracts, followed bysequencing of the associated DNA fragments(211). To overcome the low abundance of re-combination intermediates in wild-type mice, aHop2−/− mutant, which lacks a strand invasionaccessory protein and accumulates DMC1- andRAD51-bound intermediates, was first used(191). Further protocol optimization enabledrobust detection of DMC1-bound sites in wild-type mice as well (116). Approximately 10,000hotspots were identified with a p <10−4 and afalse-discovery rate of 6.7% (Figure 5). Mosthotspots are 60–330 kb apart and show a widerange in activity. Hotspots do not correlate withtranscription promoters and are located both ingenic and intergenic regions. Extending this ap-proach to female meiosis is extremely difficultbecause of the low amount of material that canbe recovered from embryonic ovaries, which iswhere meiosis occurs. However, genetic analy-sis allowed separate monitoring of male and fe-male hotspot activity, showing a large numberof hotspots with similar activity in both sexesand allowing the identification of a subset ofhotspots with significant differences in activity(178, 179).

One key determinant of hotspot location inthe mouse is the PRDM9 protein, which con-tains both a methyltransferase domain and asequence-specific DNA-binding domain com-posed of several C2H2 zinc fingers (see side-bar, PRDM9) (Figure 5) (12, 160, 183). Al-though PRDM9 binding to chromatin in mei-otic cells has not been directly demonstrated,several studies have found that mouse (and alsohuman; see below) hotspots are near predicted

PRDM9 binding sites (12, 34, 87). Moreover,two mouse strains that express two differentPrdm9 alleles showed no overlap in their DSBhotspot distributions, indicating that the posi-tion of most hotspots is determined by PRDM9DNA-binding specificity (34). This observationimplies that SPO11 is somehow recruited tothe PRDM9 binding sites and not to regionsof accessible chromatin, as it is in yeast. (87).PRDM9 has a methyltransferase domain thatpromotes H3K4me3 formation (93), and DSBsites are enriched for H3K4me3 (35, 211). Anal-ysis at one mouse hotspot showed that a singlenucleotide change within the PRDM9 bindingmotif located within this hotspot resulted in alower affinity for PRDM9 and a parallel de-crease in H3K4me3 and recombination activity,consistent with a role for PRDM9 in H3K4me3formation at hotspots (87). However, the quan-titative measures of H3K4me3 enrichment in-dicate that it is present at much lower levels atDSB hotspots than it is at transcription startsites (TSSs) (Figure 5) (35, 211). This suggeststhat, in mice, PRDM9 and/or its binding sitesmight combine two properties: recruitment ofSPO11 and the deposition of a chromatin mark.Whether H3K4me3 plays a role similar to thatdescribed in S. cerevisiae, where it is involvedin recruitment of DSB sites to the chromo-some axis (see above), is unknown. What isknown is that DSB locations are dramaticallyaltered in mutant mice lacking PRDM9: DSBformation still occurs but at new locations thatare often near H3K4me3-enriched TSSs thatare expected to be accessible chromatin regions(Figure 5) (34). These DSBs are inefficientlyrepaired, either because of their localization or apotential role for Prdm9 in DSB repair (92, 93).

A distinct control may operate in the PAR,where DSBs do not change location in strainsthat express distinct Prdm9 alleles and whereDSB hotspots are not affected by the absenceof PRDM9 (34).

Crossover Hotspot Mapsin Homo sapiens

DSB sites have not been directly mapped inhumans. Most data regarding potential DSB

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Figure 5Examples of hotspot distribution in mammals. (a). Mapping of crossover (CO) hotspots in humans. CO activity was tested in severalintervals within a 200-kb region by allele-specific polymerase chain reaction (PCR) on sperm DNA, and six hotspots of variableactivities were revealed (from Reference 104). (b) Mapping meiotic double-strand breaks (DSBs) in mice. By ChIP of DMC1 frommouse testis chromatin, followed by NGS (next-generation sequencing), DSBs were mapped and compared with H3K4me3enrichment. DSB sites show a relatively low level of H3K4me3 compared with transcription start sites (TSSs). In the absence of Prdm9,DSBs often occur near TSSs and transcription enhancers. Schematic representation adapted from Reference 34. (c) Domains of mousePRDM9. PRDM9 contains a KRAB (Kruppel-associated box) domain, which is potentially involved in protein-protein interactions(20), an SSXRD domain present in SSX proteins and involved in transcription repression (131), a PR/SET domain withmethyltransferase activity (93) and flanked by zinc fingers, and a tandem array of C2H2 zinc fingers.


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PRDM9

PRDM9 is a member of the PRDM protein family, with sev-eral members shown to be transcriptional regulators. All mem-bers have a PR domain (PRD1-BF1 and RIZ) and C2H2 zincfingers (with the exception of PRDM11, which lacks zinc fin-gers) (75, 78). The PR domain is distantly related to the SET(suvar3–9, enhancer-of-zeste, trithorax) methyltransferase do-main. PRDM9 catalyzes trimethylation of histone H3 lysine 4(93). In primates, a gene duplication gave rise to a pair of par-alogs: PRDM7 and PRDM9. PRDM9 is the only PRDM mem-ber that contains a KRAB (Kruppel-associated box) domain (20).It also contains an SSXRD domain (131). In PRDM9, the zincfinger domain is a tandem array of C2H2 zinc fingers. A PRDM9zinc finger domain is under strong positive selection at residuesinvolved in direct contact with DNA (177, 192, 227, 244). In sil-ico predictions of the DNA-binding specificity can be performed(187, 188).

Prdm9 has a meiotic specific expression and is essential formale and female fertility in mice (93). Prdm9 is also involvedin hybrid sterility in mice. Genetic incompatibilities involvingPrdm9, X-linked, and autosomal loci lead to defects during mei-otic prophase (19, 66, 154).

Linkagedisequilibrium(LD)-based hotspot:the detection ofcrossover activitybased on LD analysis.LD is a measure ofwhether alleles at twoloci coexist in apopulation in anonrandom fashionand provides a value ofsex-averaged historicalcrossover activity

hotspots come from mapping CO hotspotsthrough various methods. As in mice, thesehotspots appear to be determined by the bind-ing specificity of PRDM9. The first evidencefor this came from the identification of a DNAmotif present at 40% of linkage disequilibrium(LD)-based hotspots that shows similarity tothe predicted PRDM9 binding sequence (160)as well as from correlations between Prdm9genotypes and CO localization in pedigreeanalysis (12). These findings were extended tohotspot analysis in human populations with dif-ferent Prdm9 alleles (95), association studiesthat illustrated the role of PRDM9 in CO lo-calization (74, 122), and high-resolution anal-ysis of specific hotspots where both COs andNCOs were analyzed (15, 16). Strikingly, all18 hotspots tested for activity by sperm typ-ing are activated by specific PRDM9 variants.At a few hotspots, specific polymorphisms lo-cated within the predicted PRDM9 binding se-

quence have been correlated with variation inhotspot activity (16). Some questions remain,particularly because several hotspots show dif-ferent activity in individuals that carry differentPrdm9 alleles but do not contain DNA motifswith significant similarity to predicted PRDM9binding sites. One current limit to interpretingthese observations is that current understand-ing of the DNA-binding specificity of PRDM9zinc fingers, and of other factors that could in-fluence this specificity, is limited.

The overall view, based on population diver-sity analyses, is that the human genome containsapproximately 23,000 recombination hotspotsthat are 1–2 kb in size, are spaced approxi-mately 50–100 kb apart, display variable inten-sities, and are located in genic and intergenicregions, with a bias for being distant from TSSs(51, 159). Thus, qualitatively, human hotspotsshow similar properties to those described inmice. Sex-dependent differences in the activi-ties of some hotspots have been observed (122),but the basis of these differences is unknown.It should be noted that, unlike in mice, almostall of the information we have about hotspotsin humans is based on CO activity alone. Asis outlined in the section From Formation toRepair of DNA Double-Strand Breaks: Out-line and Main Players, meiotic DSB repair hastwo major outcomes (CO and NCO), and theCO map need not be a direct equivalent of aDSB map. Indeed, it is important to note thatlarge variation (up to 40-fold) in CO:NCO ra-tios between human hotspots has been reported(98), and the factors that influence these ratiosare not well understood. Genome-wide geno-typing or sequencing of single sperm cells hasbeen recently described in two studies, and thisnovel approach creates new possibilities for themonitoring of CO and NCO activities duringhuman spermatogenesis (138, 239).

An LD-based map of hotspots in chim-panzees (9) showed a good conservation of COactivity with humans at a broad scale (severalMb window size) but no significant overlap be-tween hotspot distributions at a finer resolu-tion. This is consistent with the divergence be-tween humans and chimpanzees within the zinc

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GC–biased geneconversion (gBGC):a process wherebymeiotic recombinationleads to an increase inGC content amongproducts

SNP:single-nucleotidepolymorphism

finger array of the various Prdm9 alleles. How-ever, overall CO activity profiles around genesand CpG islands are similar in humans andchimpanzees, suggesting a role for chromatinorganization in DSB activity. Unlike in hu-mans, regions around predicted PRDM9 bind-ing sites in chimpanzees do not show detectableincreases in recombination activity (9). This isprobably due to the limitation of predictingPRDM9 binding sites in silico.

Crossover Hotspot Map in Canisfamiliaris: SPO11 Loses ItsGuide in Canidae

Searches for PRDM9 conservation amongmammals led to the surprising finding thatPRDM9 is probably nonfunctional in Canis lu-pus familiaris. The only copy of PRDM9 in thedog genome appears to be a pseudogene, andphylogenetic analysis suggests that PRDM9,conserved in vertebrates, was lost in the canidlineage (192). This was confirmed by furtherphylogenetic studies indicating that PRDM9inactivation occurred sometime between 7 and49 Mya, predating dog domestication and caniddiversification (10, 156). Hotspot distributionwas determined by population diversity anal-ysis, and approximately 4,000 hotspots weremapped in regions of 18 kb in size or larger, theprecision of mapping being limited by low poly-morphism density. These hotspots, which arelikely to represent a subset (estimated at 10%)of all hotspots, are strikingly different from hu-man hotspots. Hotspot locations show, at best,a weak tendency to be less abundant withingenes, but show a strong enrichment for GCcontent. Using pandas and cats as outgroups todetermine substitution patterns, these hotspotregions were characterized as having a strongsubstitution bias toward GC. This GC enrich-ment, which is much stronger than in humanhotspots, might be due to GC-biased gene con-version (gBGC) (65), and its extent indicatesthat it took place over long periods. This sug-gests that, unlike recombination hotspots in hu-mans, dog hotspots are relatively stable in time(10).

Crossover and Noncrossover Hotspotsin Arabidopsis thaliana

Evidence for highly localized recombinationactivity in A. thaliana has come from several ap-proaches, including progeny analysis, genotyp-ing, and, more recently, high-throughput se-quencing or direct molecular analysis of pollenand of population diversity. Progeny analysisfirst showed that small, several-kb-long regionshave a higher rate of recombination than the av-erage of flanking sequences (64). In a study inwhich meiotic recombination was detected byhigh-throughput sequencing of the four prod-ucts of a single meiosis, both COs and NCOswere detected. COs were detected at the ex-pected rate from the genetic map, but only oneto three NCOs per meiosis could be identified,which is much lower than expected from thecounts of RAD51 or DMC1 foci (Table 1).A significant fraction of NCOs may be un-detectable in this analysis if gene conversiontracts do not include single-nucleotide poly-morphisms (SNPs), which depends on SNPdensity and gene conversion tract length (137).In contrast, by high-throughput sequencing ofF2 progeny from a cross with the same SNPdensity as the previous study, a large numberof COs and also of NCOs could be detectedat high resolution. An unexpectedly high fre-quency of COs, with many double COs (i.e.,two nearby COs in the same meiosis), was ob-served in pericentromeric regions, suggestingthat repeated sequences may interfere with theanalysis. The high number of NCO eventsdetected (from 265 to more than 3,000 permeiosis, depending on the estimation method),which may be consistent with the cytologicaldata, is however in contradiction with the otherstudy (137) and remains to be validated (252).

In a study that used LD analysis of 19 Ara-bidopsis accessions, 260 CO hotspots were iden-tified that were typically 1–2 kb wide, witha CO activity 200 times stronger than thegenome-wide average. These hotspots showeda slight preference for location outside genes(118) but determinants for their localizationare unknown. This tendency toward hotspot


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localization in intergenic regions was con-firmed in a large-scale population study thatalso revealed a higher recombination ratewithin transposable elements (99). Hotspotshave been identified in other plant speciesby genotyping, specifically near the Hga1 andHga3 genes (200) in wheat, and within the a1-sh2 region (253) and at the bz gene in maize(63).

Crossover and Noncrossover Hotspotsin Drosophila melanogaster

The pioneering study of meiotic recombina-tion events at the rosy locus of D. melanogasterled to the proposal that initiation may takeplace at multiple locations in the region stud-ied (47). This conclusion may actually rep-resent a general feature of the distributionof meiotic recombination in D. melanogaster:A genome-wide, high-resolution map of COsand NCOs in D. melanogaster was generatedby progeny analysis using high-throughput se-quencing (48). Overall, among more than 5,800meioses, 32,511 COs, and 74,453s NCO wereidentified. Interestingly, NCOs showed a muchmore even distribution than did COs, andNCOs were detected in regions with low orno CO activity, such as telomeres, centromeres,and the heterochromatic chromosome 4. Thisresult re-emphasizes that DSB activity can-not be directly extrapolated from a CO map.Among the events that could be mapped athigh resolution (+/− 500 bp, 5% of all events),NCOs showed a slight tendency to be locatedin genes (70% instead of the 60% expectedin the absence of bias) but were not closeto TSSs. In contrast, COs did not show anybias for genic or intergenic regions. Overall,these data suggest that DSB activity can oc-cur at many sites distributed relatively homo-geneously along the genome. A large numberof heterogeneous DNA motifs were identifiedas being enriched in hotspot regions, but thesignificance of these motifs remains to be an-alyzed. A study of CO distributions in a 2-Mbregion, among selected recombinant progeny,

also revealed large variation in CO activity(209).

MEIOTIC RECOMBINATION ATSPECIFIC GENOMIC REGIONS

One major challenge created by the pro-grammed induction of DSB formation duringmeiosis is the need to ensure the proper repairof all DSBs, with a minimum of deleteriousevents (deletions, duplications, and transloca-tions) that compromise genome integrity. Thepotential link between meiotic recombinationand loss of genome integrity, particularly thoseevents associated with human diseases, has beenreviewed (203). Recombination between re-peated sequences can frequently lead to suchloss of integrity. However, measuring recombi-nation in highly repeated regions is quite chal-lenging because of difficulty in identifying reli-able and well-mapped markers.

Centromeres

In organisms in which repeated sequenceseither flank or are within centromeres, COswithin centromeric regions are extremely rareor undetectable. We currently do not knowif this is due to a lack of DSBs or to a specificchanneling of DSB repair toward NCO orintersister recombination. In S. pombe, pericen-tromeric and centromeric chromatin is silencedby RNA-interference (RNAi) pathways, andseveral components of these pathways arerequired for the repression of DSB formationat these regions (68). Rec12 (Spo11) binding tothe centromeric core region of S. pombe chro-mosomes was detected in one assay in whichRec12 was cross-linked to chromatin in rad50Sstrains, but not in other experiments per-formed without cross-linking (54, 139). ChIPexperiments performed with cross-linking haveshown that S. cerevisiae Spo11 is transientlyenriched in a 20–30-kb region around thisorganism’s 120-bp-long centromeres at thebeginning of meiotic S phase, but Spo11then relocalizes to sites on chromosome arms

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Long terminal repeat(LTR): repeatedregions located at bothends of sometransposable elements

during prophase (123). Whether Spo11 has anactivity in this pericentromeric region remainsto be determined. DSBs are detected nearcentromeres but at frequencies that are reducedrelative to the genome-wide average (180).Recombination repression in S. cerevisiae peri-centromeric regions appears to be mediated,at least in part, by the Zip1 protein, a synap-tonemal complex component that localizesat centromeres at the onset of prophase andbefore DSB formation (166, 219). In zip1mutants, levels of meiotic COs and NCOs areincreased in pericentromeric regions, whereasDSB levels remain as they are in wild-type cells(44). This finding suggests that Zip1 proteinmay somehow direct DSBs that form near thecentromere toward intersister and away frominterhomolog recombination.

Telomeres

Analysis of recombination in telomeric regionsis inherently difficult because of the presenceof repeat sequences and the limited availabilityof markers. Direct measures of DSBs in S. cere-visiae reveal a partial repression of DSBs (3.5–6.5-fold reduction relative to the genome-wideaverage) in sequences within 20 kb of telomeres(23, 36, 180); the regions affected tend to begene-poor and heterochromatic as well as con-tain several classes of repeat sequences. This re-duction in DSB levels is correlated with a corre-sponding reduction in COs. Interestingly, onestudy reported increased DSBs, relative to thegenome as a whole, in the flanking regions 20–100 kb from telomeres (44), although this resultwas not observed in two other genome-widestudies (37, 180). Elevated COs in a similar re-gion were observed in genetic studies of recom-bination on a single yeast chromosome (11).Increased CO frequencies in the subtelomericregions have also been observed during malemeiosis in several mammals (106, 121) as wellas in plants (80). These regional effects couldbe related to properties of nuclear architecture,chromosome organization, and/or chromoso-mal positions within the nucleus that might be

influenced by interactions between telomeresand the nuclear envelope.

Other Repeats

Ribosomal DNA (rDNA) genes are usuallyfound in a major repeated DNA cluster andare composed of tandemly repeated genes witha specific chromatin conformation and nuclearorganization. In S. cerevisiae, DSBs are almostabsent from the 1-Mb rDNA gene cluster (23),and this repression depends on the histonedeacetylase Sir2 (153). Interestingly, the edgesof this region present a challenge for cells be-cause of the high risk of genome rearrange-ment, and DSB formation appears to be reg-ulated in these regions by a specific mechanismthat involves the AAA+ ATPase Pch2 (231).In addition, nontandem repeated sequences inthe yeast genome, such as retrotransposable el-ements of the TY family and their long terminalrepeats (LTRs), have very low (but detectable)DSB activity (180). This partial suppression isassociated with a closed chromatin configura-tion, at least in the Ty element studied (14).

An unexpected result from high-resolutionmapping of human hotspots was the detec-tion of hotspot activity within the THE1A andB families of retrotransposons and an over-representation of their LTRs among hotspots(159). Specifically, 10% of hotspots contain,within a repeat element, a perfect match to theCCTCCCTNNCCAC sequence that is a bind-ing site for the major human PRDM9 variant.This feature is not a general property of repeatDNA given that L1 elements are underrepre-sented among hotspots (149, 161). Given thepostulated mechanism of action of PRDM9 inhotspot specification through sequence-specificDNA binding, hotspot activity in the THE1repeat family is expected to be restricted toindividuals carrying Prdm9 alleles that recog-nize the core motif mentioned above. Indeed,hotspots maps derived from African popula-tions, which contain different Prdm9 alleles, donot show increased activity at THE1 elements,and little evidence was found in these maps for


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increased recombination activity in repeated el-ements, except for a weak activity at the repeatL1PA10 and L1PA13 from the L1 family (95).

HOTSPOT DYNAMICSAND EVOLUTION

DSB repair involves the directional transferof genetic information from the noninitiatingchromatid to the initiating chromatid. Thistransfer bias, which has been demonstratedin fungi and in mammals, is thought to bedue, in part, to directionality of the mismatchrepair machinery that results in alleles thatare present on the initiating chromatid andclose to the DSB being converted to the otherparental genotype (190). This biased conver-sion can occur in both NCO and CO path-ways, as illustrated in Figure 1. Thus, allelesthat stimulate DSB activity in cis should grad-ually be eliminated by this process, leading tothe loss of DSB activity. The continued pres-ence of DSB activity, when, according to thishypothesis, it should have been eliminated overtime, is often referred to as the hotspot para-dox (33, 49). One way out of this theoreti-cal paradox is to make DSB activity indepen-dent of the DNA sequences that are includedin conversion tracts. However, in humans andmice, because recombination is promoted bysequence-specific binding of PRDM9, and be-cause PRDM9 binding sites are predicted tobe close to initiation sites and thus to be fre-quently converted, the paradox is predicted tohold. In humans and mice, several sequencechanges near hotspot centers, located withinpredicted PRDM9 binding sites, have beenshown to affect initiation activity. These SNPsare included in conversion tracts and displaythe expected transmission bias (15, 16, 87, 105).Thus, PRDM9 high-affinity binding sites areexpected to decrease in frequency throughoutthe genome during evolution. This predictionis supported by comparison of chimpanzee andhuman hotspots, which show an erosion of the13-nucleotide motif related to the binding siteof the major PRDM9 variant in the human pop-ulation (160).

A potential answer to the hotspot paradoxin mammals may involve changes in PRDM9DNA-binding specificity. Prdm9 is among thefastest evolving genes in the human genome(177, 192, 227), and PRDM9 zinc fingerresidues that are important for DNA sequencebinding specificity are under concerted and pos-itive selection. This zinc finger domain is con-tained in a minisatellite-like tandem repeat ar-ray within a single exon, and it thus has a highpotential for recombination between repeats. Avery high level of Prdm9 diversity has been re-ported in humans (12, 15, 16, 122, 177, 183),rodents (12, 111, 177, 183) and chimpanzees(9, 88), with variations in the number and iden-tity of repeats. The human PRDM9 zinc fin-ger array has been shown to be genetically un-stable, with frequent remodeling of the arrayby mitotic and meiotic recombination (103).This high mutation rate predicts that changesin hotspot usage may also occur rapidly, andthis may actually resolve the hotspot paradoxbecause the formation of mutations suppress-ing hotspot activity in cis would be slower thanchanges of hotspot usage due to Prdm9 instabil-ity (103). However, it remains to be determinedhow these dynamic properties impact hotspotlifespan and recombination activity, and howselection actually acts on Prdm9.

On the basis of calculations of changes inGC content over evolutionary time, it was pro-posed that hotspots in the dog genome may bemore stable than hotspots identified in humans.Given the absence of Prdm9 in Canidae (seeabove), it is possible that in dogs, the locationand activity of hotspots are not determined bythe local DNA sequence of sites where DSBsand gene conversion occur. If properties suchas DNA accessibility are sufficient to determinehotspot activity or if DNA motifs play a minorrole, as in S. cerevisiae, the impact of the dispar-ity of gene conversion should be very weak orabsent at dog recombination hotspots. To eval-uate these suggestions, it will be important tohave more direct information on hotspot sta-bility in Canidae.

In other organisms, the NDRs wherehotspots occur may also have other roles,

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particularly in gene expression, and thus beunder selection. In this regard, a compar-ison between S. cerevisiae meiotic recom-bination maps and Saccharomyces paradoxus

LD-based recombination hotspots indicatesthat hotspot activity is significantly conservedamong widely diverged Saccharomyces species(229).

SUMMARY POINTS

1. Spo11 is an evolutionarily conserved protein that catalyzes meiotic DSB formation.

2. DSB formation requires additional proteins whose activities are not yet characterized.Two of them, Rec114 and Mei4, are conserved in several species and localized on meioticchromosomes axes.

3. HORMA domain–containing proteins are components of meiotic chromosome axes,play important roles in DSB formation and repair, and are suggested to be involved inpositive and negative regulation of DSB formation.

4. Sites of DSB formation are determined by several layers of control: (a) Accessibility forSpo11. This accessibility depends on local properties of DSB sites, such as chromatinstructure and binding of transcription factors, and may also be influenced by chromosomeorganization. In mammals, an alternative mechanism involving the DNA-binding proteinPRDM9 appears to promote the recruitment of Spo11. (b) DSB sites should also bein sequences that associate with the chromosome axis before or at the time of DSBformation. Several proteins mediate or stabilize these interactions, with two importantmediators being Spp1 in S. cerevisiae and Mde2 in S. pombe.

5. One consequence of the mechanisms involved in DSB formation is a highly punctuatedpattern of DSB localization. Whether this is the case in all organisms remains to bedetermined. The role of DSBs in stabilizing homologous interactions in several organ-isms probably requires DSB formation at multiple sites with a broad distribution alongchromosomes.

6. Hotspot locations appear to be highly dynamic in mammals, a consequence of the rapidevolution of the DNA-binding domain of PRDM9. This indicates a great flexibility in thelocalization of meiotic DSB events. The evolutionary constraints on hotspot localizationand the consequences of these dynamic changes on genome evolution remain to beunderstood.

FUTURE ISSUES

1. The molecular machinery for DSB formation remains to be characterized, starting withthe enzymatic activity of Spo11, obviously a key point to clarify. The other proteinsinvolved may have regulatory or structural roles. Insight might thus be gained throughthe identification of this regulation, possibly involving different protein modificationsand interactions with chromosome axis proteins.

2. How DSB formation is regulated in time and space still remains to be understood. Insightinto this process may be gained from understanding of the role of ATM and other DNAdamage-response kinases in regulating DSB formation.


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3. The available high-resolution methods (single cell– or population-based) are extremelyuseful, but ideally new strategies should be developed to follow the dynamics of DSBformation and the interaction of DSB sites with the chromosome axis. Insight into theorganization of chromosome loops and the chromosome axis is essential for furtherprogress on the mechanisms and regulation of DSB formation and meiotic recombinationin general.

4. The consequences of hotspot localization for genome evolution remain unclear, both atthe molecular and evolutionary levels. What is the significance of DSBs being close toor far from regions involved in gene expression?

5. It has been difficult to evaluate the impact of gene conversion events on genome dynamicsbecause of the limited data available documenting NCO events in most species. Next-generation sequencing approaches are providing the tools to obtain an entirely new set ofdata regarding meiotic recombination events. The evolutionary consequences of havingstable or dynamic DSB sites remain to be understood.

6. Although the focus of this review has been on the molecular mechanisms and propertiesthat are required for normal recombination activity, mechanisms may exist that preventmeiotic recombination from occurring in specific circumstances, e.g., in the preventionof recombination between nonhomologous or partially homologous chromosomes. Thisissue can be addressed from a mechanistic point of view but also from an evolutionarypoint of view given that recombination barriers caused by genetic divergence may providea link between recombination and speciation.

DISCLOSURE STATEMENT

The author is not aware of any affiliations, memberships, funding, or financial holdings that mightbe perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

I thank Mathilde Grelon and Jerome Buard for critical reading of the manuscript and all membersof my laboratory for discussions about the various topics presented in this review. My group isfunded by grants from CNRS, ANR (ANR-09-BLAN-0269-01), and FRM.

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GE47-FrontMatter ARI 2 November 2013 9:9

Annual Review ofGenetics

Volume 47, 2013

Contents

Causes of Genome InstabilityAndres Aguilera and Tatiana Garcıa-Muse � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Radiation Effects on Human HeredityNori Nakamura, Akihiko Suyama, Asao Noda, and Yoshiaki Kodama � � � � � � � � � � � � � � � � � � �33

Dissecting Social Cell Biology and Tumors Using Drosophila GeneticsJose Carlos Pastor-Pareja and Tian Xu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �51

Estimation and Partition of Heritability in Human Populations UsingWhole-Genome Analysis MethodsAnna A.E. Vinkhuyzen, Naomi R. Wray, Jian Yang, Michael E. Goddard,

and Peter M. Visscher � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �75

Detecting Natural Selection in Genomic DataJoseph J. Vitti, Sharon R. Grossman, and Pardis C. Sabeti � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �97

Adaptive Translation as a Mechanism of Stress Responseand AdaptationTao Pan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 121

Organizing Principles of Mammalian Nonsense-MediatedmRNA DecayMaximilian Wei-Lin Popp and Lynne E. Maquat � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 139

Control of Nuclear Activities by Substrate-Selectiveand Protein-Group SUMOylationStefan Jentsch and Ivan Psakhye � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 167

Genomic Imprinting: Insights From PlantsMary Gehring � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 187

Regulation of Bacterial Metabolism by Small RNAsUsing Diverse MechanismsMaksym Bobrovskyy and Carin K. Vanderpool � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 209

Bacteria and the Aging and Longevity of Caenorhabditis elegansDennis H. Kim � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 233

The Genotypic View of Social Interactions in Microbial CommunitiesSara Mitri and Kevin Richard Foster � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 247

SIR Proteins and the Assembly of Silent Chromatin in Budding YeastStephanie Kueng, Mariano Oppikofer, and Susan M. Gasser � � � � � � � � � � � � � � � � � � � � � � � � � � � � 275

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GE47-FrontMatter ARI 2 November 2013 9:9

New Gene Evolution: Little Did We KnowManyuan Long, Nicholas W. VanKuren, Sidi Chen, Maria D. Vibranovski � � � � � � � � � � � 307

RNA Editing in Plants and Its EvolutionMizuki Takenaka, Anja Zehrmann, Daniil Verbitskiy, Barbara Hartel,

and Axel Brennicke � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 335

Expanding Horizons: Ciliary Proteins Reach Beyond CiliaShiaulou Yuan and Zhaoxia Sun � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 353

The Digestive Tract of Drosophila melanogasterBruno Lemaitre and Irene Miguel-Aliaga � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 377

RNase III: Genetics and Function; Structure and MechanismDonald L. Court, Jianhua Gan, Yu-He Liang, Gary X. Shaw, Joseph E. Tropea,

Nina Costantino, David S. Waugh, and Xinhua Ji � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 405

Modernizing the Nonhomologous End-Joining Repertoire:Alternative and Classical NHEJ Share the StageLudovic Deriano and David B. Roth � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 433

Enterococcal Sex Pheromones: Signaling, Social Behavior,and EvolutionGary M. Dunny � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 457

Control of Transcriptional ElongationHojoong Kwak and John T. Lis � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 483

The Genomic and Cellular Foundations of Animal OriginsDaniel J. Richter and Nicole King � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 509

Genetic Techniques for the ArchaeaJoel A. Farkas, Jonathan W. Picking, and Thomas J. Santangelo � � � � � � � � � � � � � � � � � � � � � � � 539

Initation of Meiotic Recombination: How and Where? Conservationand Specificities Among EukaryotesBernard de Massy � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 563

Biology and Genetics of Prions Causing NeurodegenerationStanley B. Prusiner � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 601

Bacterial Mg2+ Homeostasis, Transport, and VirulenceEduardo A. Groisman, Kerry Hollands, Michelle A. Kriner, Eun-Jin Lee,

Sun-Yang Park, and Mauricio H. Pontes � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 625

Errata

An online log of corrections to Annual Review of Genetics articles may be found athttp://genet.annualreviews.org/errata.shtml

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Documents

Initiation of meiotic recombination how and where Conservation and specificities among eukaryotes_DeMassy_2013