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

GE47CH24-deMassy ARI 29 October 2013 15:11
Initiation of Meiotic
Recombination: How and
Where? Conservation and
Specificities Among
Eukaryotes
Bernard de Massy
Institute of Human Genetics, Centre National de la Recherché Scientifique, UPR1142,
34396 Montpellier, France; email: bernard.de-massy@igh.cnrs.fr
Annu. Rev. Genet. 2013. 47:563–99
The Annual Review of Genetics is online at
genet.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, sexual
reproduction
Abstract
Meiotic recombination is essential for fertility in most sexually repro-
ducing species. This process also creates new combinations of alleles
and 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 are
catalyzed by the evolutionarily conserved SPO11 protein, assisted by
several other factors. Some of them are absolutely required, whereas
others are needed only for full levels of DSB formation and may par-
ticipate in the regulation of DSB timing and frequency as well as the
coordination between DSB formation and repair. The sites where DSBs
occur are not randomly distributed in the genome, and remarkably dis-
tinct strategies have emerged to control their localization in different
species. Here, I review the recent advances in the components required
for DSB formation and localization in the various model organisms in
which these studies have been performed.
563
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Homologous
chromosomes
(homologs): the two
parental chromosomes
present in a diploid cell
Sister chromatids:
the two chromatids
that result from a
round of DNA
replication
INTRODUCTION
In the sexual reproductive cycle, diploid cells
are converted into haploid cells through meio-
sis, in which a single round of DNA repli-
cation is followed by two divisions. During
the first meiotic division, homologous chromo-
somes (homologs) segregate, and in the second
division, sister chromatids segregate. The
first segregation, called reductional segrega-
tion, faces a situation unique to meiotic cells,
whereby the two homologs must identify each
other and segregate to the opposite poles. This
involves 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 1
Connecting homologs by recombination. Meiotic double-strand breaks (DSBs) are induced at the beginning
of 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 in
part by controlling the localization of the involved proteins (81). Ectopic interactions do occur but are
counteracted 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 as
noncrossovers (NCOs) or by recombination with the sister chromatid, with the latter occurring particularly
in cases where homologs do not share homology at the site of the DSB (82) and for heterogametic sex
chromosomes. During interhomolog CO or NCO recombination, DNA sequences from the initiating
chromatid (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 homologs
find each other and establish connections in or-
der to orient properly at the metaphase of the
first meiotic division.
In most species, these connections are real-
ized by reciprocal homologous recombination
events, also called crossovers (COs), and visu-
alized at the cytological level as chiasmata. One
CO per homolog pair is sufficient to hold them
together in collaboration with the coordinated
establishment of cohesion between sister chro-
matids (Figure 1). In some species, such as in
Drosophila melanogaster males, the homolog pair
564 de Massy

Crossover (CO):
a recombination that
leads to the exchange
of genetic markers
flanking the crossover
point
Chiasmata:
the cytological
manifestations of a
crossover
Gene conversion:
recombination that
results in
nonreciprocal transfer
of genetic information
from one DNA duplex
to another
Noncrossover
(NCO):
a recombination event
that is detected as a
gene conversion event
without the exchange
of flanking markers
Double-strand break
(DSB): a DNA
molecule with both
strands broken at the
same position or at
closely spaced
positions
Double-strand break
repair (DSBR): the
process of repairing
DSBs. In meiosis,
DSBs are repaired by
homologous
recombination
is held together by a distinct mechanism that
does not involve recombination (148). The me-
chanical role of homologous recombination in
chromosome segregation and the rule of the
obligatory crossover imply a tight regulation
of 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 genetic
defects among progeny, with consequent in-
fertility (162), and genetic diseases (134, 203,
216). The frequency of meiotic homologous
recombination is several orders of magnitude
greater than that of mitotic recombination [a
relatively rare event often associated with oc-
currence of DNA lesions or DNA replication
errors of various origins (3)] and has a ma-
jor effect on genome diversity. Meiotic recom-
bination has multiple influences on genome
evolution. In particular, meiotic recombination
breaks down combinations of alleles, allowing
a 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 of
information, called gene conversion; however,
a fraction of meiotic recombination events are
gene conversions without CO, also called non-
crossovers (NCOs) (Figure 1). Gene conver-
sion also has consequences for genome diversity
that 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 pairing
to various extents in different species and thus to
proper chromosome segregation. Remarkably,
meiotic recombination occurs through an evo-
lutionarily conserved program of induction and
repair of DNA double-strand breaks (DSBs).
Several key steps and genes involved in DSB
formation and repair are conserved, although
different mechanisms and strategies for regu-
lation are observed when comparing different
species.
This review focuses on the formation of mei-
otic DSBs and covers the various players in dif-
ferent model organisms, taking into account the
most recent findings since the publications of
recent reviews on this topic (67, 114, 178). The
step of DSB formation is known as the initia-
tion step of meiotic recombination (I keep this
terminology, although this is an empirical and
somewhat arbitrary definition), and it defines
the first detected chemical modification at the
DNA level.
The review first summarizes the molecular
mechanism of meiotic recombination to high-
light the major steps of DSB formation and
repair and the most important features of their
regulation (see section From Formation to
Repair of DNA Double-Strand Breaks: Outline
and Main Players, below). However, I do not
discuss in detail the features of DSB repair and
CO control (see 255 for review). I then present
the current knowledge on the trans-acting
factors involved in DSB formation, those
essential as well as those required for full DSB
activity (see sections Essential Proteins for the
Formation of Mitotic DNA Double-Strand
Breaks, and Control and Regulation of DNA
Double-Strand Breaks, below), and on the ge-
nomic sites where meiotic recombination takes
place (see sections Where Are Double-Strand
Breaks Formed?, Meiotic Recombination
at Specific Genomic Regions, and Hotspot
Dynamics and Evolution, below). This allows
outlining of the main conserved molecular
features as well as the species-specific steps of
this mechanism and future research directions.
FROM FORMATION TO REPAIR
OF DNA DOUBLE-STRAND
BREAKS: OUTLINE
AND MAIN PLAYERS
Genetic analyses in fungi led to predictions
of the molecular mechanism of meiotic re-
combination, with various hypotheses for how
this event could be initiated, which interme-
diates were involved, and how they could be
processed. The double-strand break repair
(DSBR) model was established on the basis
of several characteristics of gene conversion
and COs in fungi and on the analysis of
www.annualreviews.org • Initiation of Meiotic Recombination 565

Topoisomerases:
enzymes that alter
DNA topology by a
reaction involving
DNA breakage and
ligation
DSB repair events in mitotic cells (224). The
uneven frequency of gene conversion along
chromosomes and evidence for gradients of
gene conversion, two key features of meiotic
recombination, suggested the existence of
speciﬁc sites that promote initiation. It was
proposed that observed cases of disparity in
gene conversion, where genetic information is
transferred preferentially from one homolog to
the other, could be explained if recombination
was initiated by a DSB that was then processed
into a double-strand gap, making the initiating
chromatid the recipient of genetic information.
The direct detection of meiotic DSBs in Saccha-
romyces cerevisiae provided the ﬁrst molecular
evidence supporting the DSBR model (39, 173,
230). Further analysis of these intermediates
led to a slight variation of the model in which
DSB is not enlarged into a gap but where only
one DNA strand on each side of the DSB
is removed (223). DSBs can be repaired by
several pathways, the usage of which can differ
between different organisms (Figure 1).
ESSENTIAL PROTEINS FOR THE
FORMATION OF MEIOTIC DNA
DOUBLE-STRAND BREAKS
Spo11: The Catalytic Activity for
Double-Strand Break Formation
Meiotic DSBs are catalyzed by the evolution-
arily conserved Spo11 protein, which is ho-
mologous to TopoVIA, the catalytic subunit
of a type II DNA topoisomerase (17, 115)
(Figure 2). Spo11 is composed of two domains:
Strand invasion
MRN, CtiP, ExoI
Spo11
removal
and end
processing
Loading of Rpa, followed
by Rad51/Dmc1
Spo11 and others…
DSB
formation
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 2
Double-strand break (DSB) formation and detection. Spo11 binds to DNA as a dimer and forms DSBs in a
process 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 are
further processed, leading to long 3 single-strand tails that are bound by Rpa and then displaced by the
strand exchange proteins Rad51 and Dmc1. These intermediates can be detected in wild-type or mutant
cells, using several enrichment strategies.
566 de Massy

Chromatin
immunoprecipitation
(ChIP): a method to
identify the DNA
sequences to which a
given protein binds
(directly or indirectly).
The protein of interest
is immunoprecipitated
from purified
chromatin and
associated DNA is
recovered and
analyzed
Pseudoautosomal
region (PAR): region
on a sex chromosome
that is homologous
between the X and the
Y chromosomes
(a) a DNA-binding core that contains a winged-
helix domain (WHD), often referred to as a
catabolite activator protein (CAP) domain be-
cause it resembles the WHD domain of a CAP,
and (b) a TOPRIM domain found in a variety of
topoisomerases and primases (7). The structure
of archaeal TopoVIA has been solved (165) and
shows that TopoVIA has a dimeric organization
in which a DSB is generated by the coordinated
action of two TopoVIA subunits.
Similar to TopoVIA, Spo11 forms a tran-
sient intermediate in which the 5 ends of DNA
are covalently linked to Spo11 through a ty-
rosine that is conserved among TopoVIA and
Spo11 orthologs (144). This critical tyrosine
has been identified and validated by mutage-
nesis in S. cerevisiae (17), in Schizosaccharomyces
pombe (41), in Arabidopsis thaliana (91) and in
Mus musculus (24, 40). TopoVIA induces DSBs
with a 2-bp 5 overhang (37), like Spo11 (133).
However, unlike the transient breaks formed
in a TopoII reaction, Spo11-induced DSBs
are not re-ligated, and thus DSB formation by
Spo11 appears to be irreversible. Nevertheless,
in some situations, Spo11 cleavage might be
reversible. Indeed, chromatin immunoprecip-
itation (ChIP) analysis of Spo11-DNA com-
plexes in S. cerevisiae has led to the detection
of intermediates compatible with a reversible
reaction (193). The removal of Spo11 by the
combined action of several proteins, such as
the MRX complex (Mre11, Rad50, and Xrs2)
in S. cerevisiae, the MRN complex (MRE11,
RAD50, and NBS1) in other species, and Sae2
in S. cerevisiae (named CTIP in other species),
drives the irreversibility of the reaction. This
is achieved by endonucleolytic cleavage of the
strand 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 by
exonucleolytic 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 amino
acids in the N-terminal region. These two iso-
forms may have distinct functions, as suggested
by the observation that male mice expressing
only SPO11β are not fully fertile. This phe-
notype correlates with a defect of DSB forma-
tion in the pseudoautosomal region (PAR) of
the sex chromosomes, whereas autosomal DSB
formation appears mostly normal. It was thus
proposed that SPO11α is required for promot-
ing late DSBs, such as those normally occur-
ring in the PAR (110). In A. thaliana, three
Spo11 paralogs have been identified, two of
which (SPO11-1 and SPO11-2) are required for
DSB 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 DSB
formation 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 for
Direct Detection of Double-Strand Breaks, for
DSB mapping methods) indicated that S. cere-
visiae Spo11 has, at most, a low level of sequence
specificity (58, 62, 133, 180, 248, 249). The
genome-wide map of DSB sites in S. cerevisiae,
obtained by sequencing Spo11-linked oligos,
showed only a slight preference for occurring
in AT-rich regions (64.6% versus 60.3% in
the 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 analysis
of the characteristics of DSB sites have been
performed in several species and are discussed
below (see section Where Are Double-Strand
Breaks Formed?, below).
Few in vitro assays have been developed to
assess Spo11 function, in part because of the
insolubility of recombinant Spo11. Recently,
the DNA-binding activity of an A. thaliana
ortholog (SPO11-1) was reported, but no DNA
cleavage could be detected (208). OsSPO11-4,
which is one of the five rice Spo11 orthologs
and which is required for meiosis, interacts
with the O. sativa TopoVIB subunit (the second
subunit of TopoVI) in yeast two-hybrid assays
and in glutathione S-transferase pull-down

METHODS FOR DIRECT DETECTION OF
DOUBLE-STRAND BREAKS
The challenge of the molecular detection of double-strand breaks
(DSBs) is twofold: DSBs are transient, and at a given location in
thegenometheytakeplaceonlyinasmallfractionofmeioticcells.
DSBs can be detected by Southern blotting with various levels
of resolution (from pulsed-field gel electrophoresis to nucleotide
resolution) with a sensitivity of 0.1% of total DNA molecules (36).
DSBs can also be detected by end labeling and radio-labeled or
fluorescent nucleotides, or by terminal transferase followed by
polymerase chain reaction amplification. These approaches have
been used in yeast (225, 248), mice (195, 211, 258), Caenorhabditis
elegans (152), and maize (185).
DSBs can be detected by ChIP of Spo11 or other recom-
bination proteins (Rpa, Dmc1, Rad51). As Spo11 is transiently
covalently bound to DNA, it can be detected by ChIP without
cross-linking.
Enrichment of DSB fragments can be achieved with the use
of 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-linked
oligonucleotides, which is the most sensitive, quantitative, and
resolutive 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 circular
DNA efficiently in vitro, both in the presence
and in the absence of TopoVIB (5). Analysis
of OsSPO11-4 carrying a mutation in the pre-
dicted catalytic tyrosine is needed to validate
these results.
Spo11 appears to have evolved as the result
of an ancient duplication in eukaryotes of an
ancestral TopoVIA gene common to eukary-
otes and archaebacteria. Strikingly, no Spo11
has been identified in Dictyostelium discoideum,
where it appears to have been lost, although a
gap in the genome sequence cannot be com-
pletely excluded (144). Finally, it should be
noted that a Spo11-independent pathway for
the initiation of meiotic recombination that en-
sures chromosome segregation was detected in
S. pombe strains with a mutation in the Rad2 flap
endonuclease. This endonuclease is required
for processing Okazaki fragments, and its ab-
sence may lead to increased levels of single-
strand nicks. These lesions, whether converted
into 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 subunit
points out the conservation of some residues
in the Spo11 N-terminal region, which, in
the archaeal orthologs, are at the interface
between TopoVIA and TopoVIB (52, 85).
Orthology searches have not identified any
Spo11-interacting subunit so far. However, a
TopoVIB subunit was identified in plants, but
it is not required for meiotic recombination (89,
90, 221, 254).
Several proteins that interact directly or
indirectly with Spo11 and are required for
meiotic DSB formation (as indicated by the
absence of DSBs in the corresponding mu-
tants) have been identified in S. cerevisiae (114)
(Table 1, Genes essential for DSB). Only some
of these Spo11-associated proteins are con-
served. Various approaches, either biochemi-
cal to detect protein interactions or cytological
to monitor nuclear localization, have been de-
veloped to analyze their properties. At the be-
ginning of meiotic prophase, a proteinaceaous
structure assembles to build chromosome axes
from which emanate chromatin loops. As pre-
sented in the section Control and Regulation
of DNA Double-Strand Break Formation, this
organization plays an important role for DSB
formation and repair. Thus, nuclear localiza-
tion of several proteins and their dynamics
during meiotic prophase has provided key in-
formation regarding their roles in meiotic
recombination. On the basis of these various ap-
proaches performed in S. cerevisiae and S. pombe,
the Spo11-associated proteins can be organized
in three different subcomplexes (Figure 3).
568 de Massy

Table 1 Genetic control of double-strand break (DSB) formationa
Saccharomyces
cerevisiae
Schizo-
saccharomyces
pombe
Arabidopsis
thaliana
Caenorhabditis
elegans
Drosophila
melanogaster
Mus
musculus Activity
DSB
number
140–170 in
dmc1Δ, 160
in wild type
150–294 in
wild type, up
to 342 in
mdn1−/−, and
381 in atr−/−
12–46 in
rad-54
14 in wild
type, up to
24 in spn-A
(rad51),
spn-B (rad51
paralog), and
spn-D (rad51
paralog)
230–400 in
males,
250–370 in
females
Method DSB
fragments
or Spo11-
oligos
DMC1 or
RAD51 foci
RAD51 foci
and Tunel
assay
γH2AV foci RAD51 and
DMC1 foci
Reference 36 202, 42, 232,
125
152, 171, 198 150, 109 See
references
in 13
Genes
essential
for 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 Protein
serine/
threonine
kinase
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 )

Table 1 (Continued )
Saccharomyces
cerevisiae
Schizo-
saccharomyces
pombe
Arabidopsis
thaliana
Caenorhabditis
elegans
Drosophila
melanogaster
Mus
musculus Activity
Genes
needed
for wild-
type
DSB
activity
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 Cohesin
complex
subunit
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
a
The estimated number of meiotic DSBs in various species with the method of detection is shown. Genes involved are displayed in two parts: those
essential 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 and
Ski8, a WD (tryptophan–aspartic acid) repeat
protein that directly interacts with residues in
the C-terminal domain of Spo11, as indicated
by yeast two-hybrid assays (8). The interac-
tion between the S. pombe orthologs of Spo11
(Rec12) and Ski8 (Rec14) was also detected by
two-hybrid assay (217) and coimmunoprecipi-
tation (CoIP) (155). Ski8 is evolutionarily con-
served, expressed in meiotic and nonmeiotic
cells, and involved in RNA processing in S.
cerevisiae mitotic cells (6). The requirement for
Ski8 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 part
of the PAF complex (involved in transcription
initiation and elongation) and of the SKI com-
plex (RNA surveillance and processing) (263).
Whether WDR61 also plays a role in meiotic
DSB formation is unknown.
The second complex is composed of Rec102
and Rec104 in S. cerevisiae (107, 112, 113, 201),
but orthologs have not been identiﬁed in other
species. These two proteins interact with each
other, and Rec104 also interacts with Spo11.
Rec102 and Rec104 bind to chromatin and are
570 de Massy

A. thaliana
Prd2
DFO
Spo11-2Spo11-1
Prd1
Prd3
M. musculus
Mei4
Mei1
Spo11Rec114
S. pombe
Rec14
Rec6
Mde2
Rec7
Rec24
Rec15
Rec12
S. cerevisiae
Rec114
Mei4
Xrs2
Rad50
Mre11
Rec104
Rec102
Ski8
Spo11
Mer2
Figure 3
Proteins that are essential for double-strand break (DSB) formation and their interactions in Saccharomyces cerevisiae, Schizosaccharomyces
pombe, Arabidopsis thaliana, and Mus musculus. Interactions were detected by coimmunoprecipitation (red arrows) and in yeast two-hybrid
assays ( gray or black arrows). The two-hybrid-based interaction map for S. cerevisiae proteins is from Maleki et al. (142), with gray arrows
indicating 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 chromatin
along chromosomes, with some preference for
the chromosome axis; however, they are not
preferentially enriched near DSB sites and their
role remains to be determined (113, 182). In
S. pombe, Rec6 has been identiﬁed as an ad-
ditional partner that is essential for meiotic
DSB formation; it forms a complex with two
other proteins, Rec12 and Rec14 (61). Rec6
does not interact with Rec12 or Rec14 indi-
vidually in yeast two-hybrid assays but does so
when they are coexpressed (155). Yeast two-
hybrid assays and CoIP experiments suggest
that the S. cerevisiae Rec102-Rec104 complex
might form a bridge between Spo11 and a
third complex (Rec114-Mei4-Mer2) (8). Sim-
ilarly, the S. pombe Rec12-Rec14-Rec6 com-
plex interacts with the S. pombe orthologs of
Rec114-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), and
Mer2 colocalizes with Rec114 and Mei4 at
chromosomal axis sites (129, 142). Mer2 is a key
control point in the function of this complex
and provides a link between DNA replication
and DSB formation. DSB formation occurs
after S phase, and although replication is

Synaptonemal
complex: the protein
structure that stably
pairs homologous
chromosome axes from
end to end during
meiotic prophase via a
process called synapsis
γH2AX: the
phosphorylated form
of the histone H2A
variant H2AX on
serine 139; detected
upon DSB formation
after activation of the
ATM/ATR kinases
not essential for DSB formation (97), DNA
replication and DSB formation are temporally
coupled (28). Mer2 is phosphorylated by two
kinases that are required for DNA replication:
Cdc28 (in association with the B-type cyclins
Cbl5 and Clb6) (94) and Cdc7 (in interaction
with Dbf4) (204, 237). Phosphorylation has
been detected at several sites, two targeted
by Cdc28 and several others by Cdc7. Some
of the residues phosphorylated by Cdc7 are
primed by Cdc28-dependent phosphorylation
at residue S30 (157). Mer2 phosphorylation
is 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 binding
to axis sites still occurs efficiently, whereas
binding of Rec114 and Mei4 is mostly lost
(182). Mer2 regulation is thus thought to
provide a link between DNA replication and
DSB formation, suggesting that other proteins
of the Spo11-associated complexes are loaded
onto chromosome axes during meiotic S phase
(28, 157). Mer2 association with axis sites
does not require Mei4 and Rec114, which is
consistent with the idea that these proteins are
recruited by Mer2 (182). Rec114-Mei4-Mer2
association with the chromosome axis is
transient and declines after DSB formation
(182).
In S. pombe, Hsk1, the ortholog of Cdc7,
may play a similar role because it is essential
for DSB formation (174) and is required for
Spo11 recruitment to DSB sites (204). The in-
teraction between S. pombe Rec24 (Mei4) and
Rec7 (Rec114) is conserved and is also needed
for DSB formation (25, 217). Recent assays
have shown that Rec24, Rec7, and Rec15 (a
possible Mer2 ortholog) also form a complex
in which Rec7 directly interacts with Rec24.
Rec15, Rec7, and possibly Rec24 are detected
both on axis and DSB sites. Rec15 recruit-
ment to DSB sites depends on Rec24 and Rec7
but not on Rec12, suggesting that the Rec7-
Rec24-Rec15 complex may recognize some
specific 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 binds
to Rec14, thus bridging the two subcomplexes
(155).
Mei4 and Rec114 are conserved in most
eukaryotes (124). Mouse MEI4 is required
for meiotic DSB formation and interacts with
REC114 (124). MEI4 is localized in multiple
and discrete foci on the axes of meiotic chro-
mosomes, independent of SPO11 (124). The
A. thaliana Mei4 ortholog PRD2 also is re-
quired for DSB formation (59). However, Zea
mays 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 in
plants, Rec114 has functions distinct from those
in fungi and mammals. In this regard, it is
intriguing 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 involved
in strand exchange in both yeast and mam-
mals (143). It is tempting to speculate that
Mei4 and Rec114 may play a role in coordi-
nating DSB formation with later steps in DSB
repair.
A few additional factors have been identified
by using genetic approaches. M. musculus Mei1
and its A. thaliana ortholog PRD1, which are
required for DSB formation (60, 130), encode
for proteins containing Armadillo repeats with
similarities to importins, but their activities
are unknown. In plants, A. thaliana PRD3
(59) and its O. sativa ortholog PAIR1 (170)
are required for DSB formation, as is the
product of the A. thaliana DFO gene (260). In
D. melanogaster, MEI-P22 is required for DSB
formation and localizes to discrete foci at
meiotic chromosomes after the formation of
the synaptonemal complex but before the ap-
pearance of the phosphorylated form of histone
H2A variant H2AV (γHis2AV, the equivalent
of mammalian γH2AX), which forms in
572 de Massy

Lateral element:
the proteinaceous
structure that defines
the chromosome axis
at the beginning of
meiotic prophase
Cohesin: a protein
complex that holds
sister chromatids
together
response to DSB formation (132, 150). Forma-
tion of MEI-P22 foci requires Trade Embargo,
a chromatin-associated protein with C2H2 zinc
fingers that is also needed for DSB formation
(126).
It should be noted that in many organisms,
DSB formation deficiencies are not detected
by direct monitoring of DSB molecules but
by cytological detection of γH2AX and/or
accumulation of DSB repair proteins, such as
RAD51 or DMC1, that bind to the single-
stranded DNA present at DSB ends. These
assays have limited sensitivity, and they cannot
distinguish defects in DSB formation from
defects in the early stages of DSB processing.
These limitations should be taken into account
when interpreting data. In this context, one re-
liable assay that has been often used to identify
mutations that lead to a loss of DSB formation
involves the suppression of the meiotic chro-
mosome fragmentation seen in DSB repair
mutants.
CONTROL AND REGULATION
OF DNA DOUBLE-STRAND
BREAK FORMATION
Role of the Chromosome Structure
and Organization
Many observations have provided insights
suggesting that the chromosome organization
plays an important role in the regulation of var-
ious features of meiotic recombination. DSB
repair is regulated in such a way to control the
choice of the template (the sister chromatid, or
the homolog) for DSB repair and to regulate
the number of COs. Meiotic chromosomes are
organized in loops anchored to the axis (called
the lateral element at the beginning of meiotic
prophase), and DSB repair takes places in the
context of the axis (264, 265). However, studies
in S. cerevisiae indicate that DSBs mostly occur
at sites not constitutively axis-associated but
located within loops emanating from the axis,
suggesting that these regions are recruited to
the axis before or at the time of DSB formation
(21, 119). As several DSB proteins (particularly
the Rec114-Mei4-Mer2 complex) are located
on the axis, this may suggest that DSBs occur
once 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 a
meiosis-specific protein that is an important
factor in DSB formation and also promotes
interhomolog rather than intersister recombi-
nation during DSB repair. Hop1 contains a
HORMA (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 that
is thought to act as an adaptor to recruit other
proteins. 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 association
with chromosomes, with Hop1-enriched do-
mains corresponding to high-frequency DSB
domains (182). The Spo11 accessory pro-
teins Rec114/Mei4/Mer2 are also enriched at
these domains, and Mer2 is not recruited
to chromosomes in the absence of Hop1
(182).
In addition to its role in DSB formation,
the Hop1-Red1 complex is also required for
activation of the Mek1 kinase, which represses
recombination between sister chromatids
(167). The Hop1-Red1, and thus the Mek1,
complex is thought to regulate the choice
between interhomolog and intersister recom-
bination, in part by counteracting the sister
chromatid recombination-promoting activity
of the cohesin Rec8 and by inhibiting the
strand transfer activity associated with the
Rad51 protein (38, 117, 168). Mutants lacking

Rec8, a meiosis-specific subunit of cohesin,
show altered DSB formation, with different
regions showing decreased, unchanged, or
increased DSB levels (21, 117, 123). Inter-
estingly, Hop1 localization is dependent on
Rec8 in some regions but not others, and
those regions where Hop1 is retained in a rec8
mutant strain also retain Rec114 association
and DSB formation. This suggests that Rec8
helps to recruit, stabilize, and/or constrain
Hop1 at some regions but not at other regions
in which Hop1 can be loaded efficiently in the
absence of Rec8 (182).
Some of these features seem to be conserved
in other species. In S. pombe, mutants lacking
the Rec8 or Rec11 cohesin components show
region-specific reductions in meiotic recombi-
nation, and DSBs are not detected in mutants
that lack Rec10, a putative ortholog of S. cere-
visiae Red1 (69). S. pombe Rec10 and Hop1 colo-
calize on chromosome axes that are called linear
elements, and their localization is altered in the
absence of Rec8 (136). Cytological studies in-
dicate that Rec10, but not Hop1 or Mek1, is
required for Rec7 (Rec114) localization to the
axis (135). This effect is likely to be mediated
through Rec15, as Rec15 is required for Rec7
localization to DSB sites, and Rec10 and Rec15
have been shown to interact in a yeast two-
hybrid assay (155). DSB formation is partially
reduced in hop1 or mek1 mutants (53% and 70%
of the wild-type DSB levels, respectively) and
is further reduced in the double mutant (22%)
(128, 199).
In M. musculus, two Hop1 orthologs have
been identified: HORMAD1 and HORMAD2
(77, 181, 243). HORMAD1 localizes to unsy-
napsedchromosomeaxesatleptoteneandisdis-
placed upon synapsis formation. In Hormad1−/−
mutants, DSB levels are reduced to 25% of the
wild-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, and
the checkpoint response (55, 243). The role of
Hormad1 may indeed be similar to that of Hop1
in S. cerevisiae, as it is required for the stabi-
lization of MEI4 foci on the chromosome axes
in mouse spermatocytes (R. Kumar, K. Daniel,
A. Toth, B. de Massy, unpublished data).
HORMAD2 is a HORMAD1 paralog with a
very similar cytological localization with slight
differences, such as its specific accumulation on
the sex chromosomes at diplotene, that sug-
gest roles distinct from those of HORMAD1.
Indeed, Hormad2−/−
mutant female mice are
fertile, and males do not show defects in DSB
formation and repair but are deficient in ATR
recruitment to and silencing of the unsynapsed
regions of sex chromosomes (242).
In C. elegans, several HORMAD-like pro-
teins have been identified, and all are struc-
tural components of the meiotic chromosome
axis (259). In particular, HTP-3 is associ-
ated with chromosome axes throughout meiotic
prophase (141) and is required for DSB forma-
tion (84). HTP-3 is also involved in cohesin
loading (206). Interestingly, HTP-3 interacts
with MRE11, thus providing a potential link be-
tween DSB formation and repair. HTP-3 also
forms a complex with HIM-3, another Hop1
ortholog that is involved in pairing and synapsis,
and potentially regulating the use of the sister
chromatids for DSB repair (53). In C. elegans, a
connection between cohesins and HORMAD-
like proteins is suggested by the mislocaliza-
tion of HTP-3 in scc3 mutants also deficient for
REC8 loading on chromosomes (184, 238). A
large number of RAD51 foci are detected in scc3
mutants, indicating that HTP-3 axis association
is not required for DSB formation (84).
The ASY1 protein of A. thaliana has limited
homology with S. cerevisiae Hop1, and DSB
formation appears to be unaltered in asy1 mu-
tants. However, the ASY1 protein may retain
Hop1 function in regulating partner choice
in recombination (202). The O. sativa ASY1
ortholog (PAIR2) has a localization pattern
similar to that of HORMAD1 and, like ASY1,
is essential for homolog synapsis, but its
involvement in DSB formation has not been
investigated (169). However, ASY3, a protein
that interacts with ASY1, is required for full
DSB levels (73). A functional interaction with
cohesins is also suggested because SWI1, a
574 de Massy

protein required for cohesion between sister
chromatids during meiosis, is also required
for DSB formation and for normal ASY1
localization (151).
The functional conservation between
mouse and C. elegans HORMAD proteins and
S. cerevisiae Hop1 is further documented by
studies of the role of TRIP3 and Pch2, two
AAA+ ATPase orthologs that are involved in
maintaining the normal distribution of HOR-
MAD/Hop1 proteins on the chromosome
axis and that are required for the pachytene
checkpoint that is triggered by failures in
homolog synapsis (18, 32, 243). S. cerevisiae
pch2 mutants are partially defective in DSB for-
mation, with different defects in mutants with
different DSB end-processing defects, thus
suggesting a regulatory role in DSB formation
that involves a feedback from DSB processing
signals (72). This issue of feedback control in
DSB formation is further discussed below (see
section Coordination Between Formation and
Repair of DNA Double-Strand Breaks).
Linking DNA double-strand break sites
to chromosome axes: the distinct strate-
gies used in Saccharomyces cerevisiae and
in Schizosaccharomyces pombe. The axis local-
ization of the Rec114, Mei4, and Mer2 proteins,
which are required for Spo11 activity, raises
the question of how DSB sites and Spo11 are
brought to the axis (Figure 4). In S. cerevisiae,
this is mediated by the Spp1 protein, a member
of the COMPASS chromatin-modifying com-
plex that interacts preferentially with promoter
regions in mitotic cells (207, 2, 213). Spp1 does
this by interacting both with the axis-associated
protein Mer2 (see above) and, through its plant
Hop1/
Rec10
Axis Axis Axis
Rec25,
Rec27,
Mug20
S. pombe
Rec15, Rec7,
Rec24, Mde2Rec8/
Rec11
?
Spo11
NDRTF?
Spp1
Hop1/
Red1
Mer2
Mei4
Rec114
Rec8
Chromatin
loops
S. cerevisiae
Spo11
NDR
TSS
Set1 complex
Pol II
?
MEI4
M. musculus
Hormad1
SPO11
PRDM9
ZnF
SET
Figure 4
Guiding Spo11 and connecting double-strand break (DSB) sites to an axis. In Saccharomyces cerevisiae, Spo11 binds to
nucleosome-depleted regions (NDRs) adjacent to transcription start sites (TSSs). DSB sites interact with the axis through Spp1. In
Schizosaccharomyces pombe, Spo11 binds preferentially to NDRs, possibly with the contribution of transcription factors (TFs), at least at
some sites. Several proteins essential for DSB formation are associated with the chromosome axis and have the ability to interact with
DSB sites. This interaction may take place well in advance of DSB formation or at the same time. In Mus musculus, PRDM9 binds to
speciﬁc sites in the genome through its zinc ﬁnger (ZnF) domain, and is hypothesized to recruit SPO11. These regions are predicted to
be tethered to an axis where MEI4 is localized but are done so through an unknown mechanism. H3K4me3 deposited either by Set1 in
S. cerevisiae or by PRDM9 in M. musculus is depicted as a magenta star.

Histone H3
trimethylated lysine
4 (H3K4me3): this
modification is known
to be enriched in
accessible chromatin,
often near
transcription start sites
and some transcription
enhancers
Nucleosome-
depleted regions
(NDRs): regions of
the genome with low
nucleosome density as
indicated by
hypersensitivity to
digestion by nucleases
Condensin: a protein
complex that plays
roles in chromosome
organization and
compaction
homeodomain (PHD) finger, with histone H3
trimethylated lysine 4 (H3K4me3), a chromatin
mark often found near nucleosome-depleted
regions (NDRs) in chromatin that are bound by
Spo11 (27). Spp1 is required for a normal level
of DSB formation, but DSBs are still detected
in an spp1 strain. In spp1 strains, DSBs occur
at very low levels at sites used in wild-type yeast
cells, but strong DSB sites that are still Mer2-
dependent occur at new positions. These new
locations have been proposed to be chromatin
loop regions with a propensity to interact with
the chromosome axis.
In S. pombe, the association between DSB
sites and chromosome axes has been recently
described by two studies that highlight several
key components (76, 155). Rec25 and Rec27
(147), two linear element components with
no 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 Rec27
may form one or several complexes, possibly
with Mug20 (147, 214). Interestingly, genome-
wide ChIP-Chip showed that Rec10 is rel-
atively uniformly distributed along chromo-
somes, whereas Rec25, Rec27, and Mug20 are
highly enriched near most DSB sites, even in
the absence of Rec12 (76). Rec25 and Rec27
thus appear to recognize some as yet unknown
feature 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-wide
DSB map (see below) suggests that H3K4me3
does 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 key
properties: (a) They are favorable substrates for
Spo11-Rec12 binding, in part because they are
in regions of nucleosome depletion (see below);
and (b) they have properties that promote their
recruitment to the chromosome axis, although
different mechanisms for recruitment appear to
be 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 DSB
formation in C. elegans. Several condensins are
present in meiotic cells, and the condensin I
complex (246) affects DSB formation. Specif-
ically, its disruption leads to increased axis
length that is correlated with a higher num-
ber and altered localization of DSBs, which
are scored as RAD51 foci and revealed by a
TUNEL assay (152). Studies in many species
have reported correlations between axis length
and CO activity, and this phenomenon has been
described in detail in humans and mice (120).
It is possible that an extended axis may lead
to smaller and more numerous loops, which
may provide more potential for DSB activity
and/or alter CO control. Proteins other than
condensins may also be involved in this regu-
lation. For instance, a correlation between axis
length and loop sizes has been shown in mice in
which Sycp3, a component of the meiotic chro-
mosome axis, and Smc1β, a meiotic-specific co-
hesion subunit, were knocked out (172). The
impact of these mutations on DSB formation
was not assessed.
Further evidence for an association between
chromosome organization and recombination
activity comes from analysis of the M. musculus
domesticus PAR of the sex chromosomes. This
region is approximately 800 kb long, which
corresponds to about 1% of the autosomal
genome, but it is the locus of an obligate CO
during male meiosis that is required for sex
chromosome segregation. DSBs occur in the
PAR at a rate, per kilobase of DNA, that is
20-fold higher than in autosomes. Moreover,
measurement of loop sizes and axis length
show a marked difference between the PAR
and autosomes: PAR loops are threefold to sev-
enfold shorter than in autosomes and are thus
predicted to be more numerous, whereas the
axis length relative to DNA content is tenfold
greater in the PAR than in autosomes (110).
If the number of active DSB sites per loop
is a limiting factor, this specific organization
may allow for a higher DSB density. Similar
variation in properties of chromosome
576 de Massy

organization on autosomes may also explain
or contribute to observed regional variations
in DSB activity, which remain largely not
understood.
Global Effects of Chromatin
Modifications
In S. cerevisiae and S. pombe, several chromatin
modifiers affect local chromatin structure at
DSB sites, with consequences for DSB forma-
tion (see below). Some modifications may also
have chromosome-wide functions and effects.
For instance, C. elegans HIM-17 (a protein with
no identified activity) is required for normal
DSB formation, and him-17 mutants show re-
duced or delayed DSB formation and histone
H3 lysine 9 (H3K9) dimethylation (196). In the
mutant, synapsis (which is DSB independent in
C. elegans) is normal, but RAD51 foci and chi-
asmata are not detected. The HIM-17 protein
localizes to chromatin in germ cells. Him-17
genetically interacts with Rb, which can mod-
ify chromatin through interactions with his-
tone modifiers. Given that him-17 is required
to maintain a repressive mark (H3K9me2), it is
not expected to be directly involved in SPO11
accessibility. Rather, the phenotype observed in
him-17 mutants may be indirect, for example,
with an inappropriate recruitment and titration
of DSB components to regions normally re-
pressed and where the mutants cannot be active
(196).
Similarly, on the basis of analysis of RAD51
foci, the C. elegans XND-1 protein is also
required 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, but
CO frequencies and RAD51 foci are reduced
only on chromosome X. The X chromosome–
specific reduction of DSBs and COs is surpris-
ing because in wild-type worms, the XND1
protein is enriched only on autosomes. The
xnd-1 mutant also shows increased histone
H2A lysine 5 acetylation, which is catalyzed
by 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 in
xnd-1 mutants could be at the level of DSB
repair.
Coordination Between Formation and
Repair of DNA Double-Strand Breaks
DSB end recognition and processing is one of
the 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 DSB
sites independent of DSB formation and Rad50.
This interaction requires all Spo11 accessory
proteins and involves the C-terminal end of
Mre11, which contains a DNA-binding domain
(26, 29). It has been suggested that Mre11 plays
a role in remodeling chromatin at DSB sites
because 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 DSB
regulation 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 DSBs
are “turned off.” A first clue as to the nature
of this regulation comes from the recent
discovery of negative feedback control on DSB
formation, mediated by ataxia telangiectasia
mutated (ATM) (127). ATM, which belongs
to the superfamily of phosphatidylinositol
3-kinase–related kinases, is recruited to DSBs
and activated by the MRN complex through
direct interaction with NBS1. ATM promotes

a variety of events in response to DSBs, in-
cluding autophosphorylation, phosphorylation
of the H2AX histone variant, and activation of
MRE11 and of the CHK2 effector kinase (101,
241). ATM also appears to repress DSB forma-
tion in mice, as evidenced by increased levels
of SPO11-oligos in Atm−/−
mutants (127).
ATM-mediated negative regulation of meiotic
DSB formation has also been observed in
D. melanogaster through the analysis of histone
H2AV phosphorylation (109). As ATM activa-
tion occurs in the vicinity of DSBs, ATM may
limit the formation of DSBs at nearby sites on
the same chromatid, on the sister chromatid,
or on the chromatids of the homolog. Studies
in S. cerevisiae suggest that DSB formation
is constrained to occur at a given site only
once per pair of sister chromatids and that
constraints also extend to the same site on the
homolog (261). The two S. cerevisiae DNA
damage-response kinases, Mec1 and Tel1
(homologs of ATR and ATM, respectively),
appear to be involved in this inhibition,
although mutants in the two kinases have
different effects, with DSB levels being slightly
reduced in mec1 and unchanged in tel1 mutants
(261).
WHERE ARE DOUBLE-STRAND
BREAKS FORMED?
Several tools have been developed to de-
tect meiotic DSBs and genome-wide high-
resolution maps of DSBs are available for
S. 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 from
CO and NCO maps that cannot be directly ex-
trapolated to DSB activity without additional
hypotheses. Below, I discuss data obtained us-
ing these various approaches, with a specific
focus on the most recent high-resolution and
genome-wide findings that provide novel in-
sights into DSB site localization.
A general observation is that recombina-
tion events are not randomly distributed. When
events are examined at high resolution (single-
nucleotide to kilobase level), hotspots and
coldspots for recombination and DSB forma-
tion are observed; when events are observed at
low 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 hotspots
and coldspots suggests highly localized proper-
ties of the genome that are favorable to DSB
activity, and the presence of jungles and deserts
suggests that features of chromosomal domains
modulate DSB activity. Progress has been made
in understanding hotspot localization, but the
control at the level of domains remains poorly
understood.
The term hotspot itself can be misleading, as
levels of DSBs or recombination at sites called
hotspots can vary over several orders of mag-
nitude, and this calls into question the criteria
used to identify a given locus as a hotspot or to
estimate the number of hotspots in the genome.
Some researchers use as comparative criteria
the recombination activity of the adjacent re-
gions, whereas others use the genome-wide av-
erage. Because the term hotspot has been so
widely used, it is retained in the rest of the
article, keeping in mind the above-mentioned
caveats.
Double-Strand Break Maps in
Saccharomyces cerevisiae: The
Opportunistic Spo11
From the first detection of meiotic DSBs
(222) to the recent high-resolution genome
maps, many laboratories have contributed
to the description of the DSB landscape in
S. cerevisiae by developing and optimizing
several approaches (see sidebar, Methods for
Direct Detection of Double-Strand Breaks).
Sequencing Spo11-oligos led to the identifica-
tion of 3,604 DSB hotspots, which are defined
as having Spo11-oligo densities approximately
twofold higher than the genome-wide average
578 de Massy

(180). A striking feature of these hotspots is
their strong correlation with transcription
promoters (88.2% overlap). DSB sites extend
over small intervals (73.4% are 50–300 bp in
size) and are located in NDRs, which is in
agreement with the earlier evidence for the
importance of chromatin accessibility at S.
cerevisiae hotspots (176, 247). Hotspots have a
wide range of activity with continuous varia-
tion, and low levels of DSBs can be detected
in non-hotspot regions, outside transcription
promoters. Spo11 has no or little DNA
sequence specificity (see above), and DNA
accessibility appears to be one of the major
features that drives Spo11 to its substrate. In
fact, DSB activity can be targeted to specific
DNA sequences by expressing a Spo11 fusion
protein that contains the Gal4 DNA-binding
domain (186). Wider hotspots tend to have
larger NDRs and also tend to have higher DSB
activity (180). Moreover, factors that modulate
chromatin features and accessibility, such as
transcription factors and chromatin modifiers
and remodelers, have been shown to influence
DSB activity (189). One additional component
that plays a major role, through binding to
H3K4me3 and H3K4me2 and interacting
with Mer2 (see above) (Figure 4), is the
protein Spp1, which appears to tether DSB
sites into the context of chromosome axes.
Each parameter analyzed independently—such
as accessibility, measured by nucleosome
occupancy or H3K4me3 enrichment—is not a
good indicator of the quantitative levels of DSB
activity (31, 180, 228). This may be explained
by the combined requirement for chromatin
accessibility and H3K4me3 enrichment.
Genome-wide detection of COs and NCOs by
genotyping tetrads has also provided a map of
recombination events that is in agreement with
the high-resolution DSB maps (145).
Double-Strand Break Maps in
Schizosaccharomyces pombe
Hotspots have been mapped in S. pombe by
ChIP of Rec12, the S. pombe Spo11 homolog
(54, 139). In one study, a rad50S mutant
was used to recover Rec12-DNA complexes
without cross-linking, whereas in another
study, performed using a wild-type strain,
chromatin was cross-linked. Of note, in
S. pombe, no difference in DSB levels was
detected in rad50S and wild-type strains
(100). The distance between DSB hotspots in
S. pombe is greater than in S. cerevisiae (1 DSB
hotspot per 65 kb on average in S. pombe, com-
pared with 1 per 3.4 kb in S. cerevisiae), and the
number of DSB hotspots is correspondingly
about 10-fold lower. In S. pombe, DSB hotspots
are not correlated with transcription promot-
ers, and instead the strongest DSB hotspots
are enriched in large intergenic regions of
3 kb or more that include clusters of NDRs, an
enrichment most pronounced at the strongest
DSB sites. Overall, most DSBs (95%) overlap
with NDRs (57). However, as in S. cerevisiae,
nucleosome depletion is not sufficient to form a
hotspot because 2,973 NDRs can be defined in
the S. pombe genome. A correlation was also ob-
served between the presence of a DSB hotspot
and the level of noncoding RNAs transcribed
from a locus, which may be due to the greater
abundance of noncoding RNAs transcribed
from large intergenic regions (236). Genetic
and molecular analyses of a few hotspots
have identified a meioses-specific change in
chromatin structure near DSB sites and have
identified roles for transcription factors in influ-
encing DSB activity. The most detailed analysis
was developed at the ade6-M26 hotspot, where
activity depends on the presence of a consensus
to the binding site of the heterodimeric tran-
scription factor Atf1.Pcr1 (220, 234). Several
chromatin modifiers and remodelers also influ-
ence DSB activity (96, 250). Complementary
to these analyses, the search for new hotspots
led to the identification of hotspot-specific
DNA motifs, some of which are recognized
by transcription factors (218, 219). On the
basis of these observations, it was hypothesized
that a family or families of transcription
factors might actually recruit Rec12, either
directly or indirectly (234, 235). No specific

enrichment for H3K4me3 is observed at DSB
sites in S. pombe, and the role of Set1, if any,
in DSB formation in S. pombe remains to be
understood (251).
Double-Strand Break Map in Mus
musculus: A Molecular Strategy to
Target Double-Strand Break Activity
Genome-wide mapping of DSB hotspots in the
mouse was recently performed by ChIP for
the recombinases DMC1 and RAD51 in chro-
matin purified from testis extracts, followed by
sequencing of the associated DNA fragments
(211). To overcome the low abundance of re-
combination intermediates in wild-type mice, a
Hop2−/−
mutant, which lacks a strand invasion
accessory protein and accumulates DMC1- and
RAD51-bound intermediates, was first used
(191). Further protocol optimization enabled
robust detection of DMC1-bound sites in wild-
type mice as well (116). Approximately 10,000
hotspots were identified with a p <10−4
and a
false-discovery rate of 6.7% (Figure 5). Most
hotspots are 60–330 kb apart and show a wide
range in activity. Hotspots do not correlate with
transcription promoters and are located both in
genic and intergenic regions. Extending this ap-
proach to female meiosis is extremely difficult
because of the low amount of material that can
be recovered from embryonic ovaries, which is
where meiosis occurs. However, genetic analy-
sis allowed separate monitoring of male and fe-
male hotspot activity, showing a large number
of hotspots with similar activity in both sexes
and allowing the identification of a subset of
hotspots with significant differences in activity
(178, 179).
One key determinant of hotspot location in
the mouse is the PRDM9 protein, which con-
tains both a methyltransferase domain and a
sequence-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 also
human; see below) hotspots are near predicted
PRDM9 binding sites (12, 34, 87). Moreover,
two mouse strains that express two different
Prdm9 alleles showed no overlap in their DSB
hotspot distributions, indicating that the posi-
tion of most hotspots is determined by PRDM9
DNA-binding specificity (34). This observation
implies that SPO11 is somehow recruited to
the PRDM9 binding sites and not to regions
of accessible chromatin, as it is in yeast. (87).
PRDM9 has a methyltransferase domain that
promotes H3K4me3 formation (93), and DSB
sites are enriched for H3K4me3 (35, 211). Anal-
ysis at one mouse hotspot showed that a single
nucleotide change within the PRDM9 binding
motif located within this hotspot resulted in a
lower affinity for PRDM9 and a parallel de-
creaseinH3K4me3andrecombinationactivity,
consistent with a role for PRDM9 in H3K4me3
formation at hotspots (87). However, the quan-
titative measures of H3K4me3 enrichment in-
dicate that it is present at much lower levels at
DSB hotspots than it is at transcription start
sites (TSSs) (Figure 5) (35, 211). This suggests
that, in mice, PRDM9 and/or its binding sites
might combine two properties: recruitment of
SPO11 and the deposition of a chromatin mark.
Whether H3K4me3 plays a role similar to that
described in S. cerevisiae, where it is involved
in recruitment of DSB sites to the chromo-
some axis (see above), is unknown. What is
known is that DSB locations are dramatically
altered in mutant mice lacking PRDM9: DSB
formation still occurs but at new locations that
are often near H3K4me3-enriched TSSs that
are expected to be accessible chromatin regions
(Figure 5) (34). These DSBs are inefficiently
repaired, either because of their localization or a
potential role for Prdm9 in DSB repair (92, 93).
A distinct control may operate in the PAR,
where DSBs do not change location in strains
that express distinct Prdm9 alleles and where
DSB hotspots are not affected by the absence
of PRDM9 (34).
Crossover Hotspot Maps
in Homo sapiens
DSB sites have not been directly mapped in
humans. Most data regarding potential DSB
580 de Massy

a
DNA
3 mcM
100
10
1
0.1
0.01
3 mcM 4 mcM
?
RING3
kb
cMMb
DMA
DMB
PSMB9
PSMB8
TAP1
TAP2
100 150 200500
0
KRAB PR/SET Zn Zn ZnZnZnZnZnZnZnZnZnZnZnZn
1 248 368 518 847SSXRD
G278
Zn
c
Genes
H3K4me3
DSB
DSB
H3K4me3
Wild type
Prdm9–/–
* *
Promoters
b
Figure 5
Examples of hotspot distribution in mammals. (a). Mapping of crossover (CO) hotspots in humans. CO activity was tested in several
intervals within a 200-kb region by allele-specific polymerase chain reaction (PCR) on sperm DNA, and six hotspots of variable
activities were revealed (from Reference 104). (b) Mapping meiotic double-strand breaks (DSBs) in mice. By ChIP of DMC1 from
mouse testis chromatin, followed by NGS (next-generation sequencing), DSBs were mapped and compared with H3K4me3
enrichment. 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 mouse
PRDM9. PRDM9 contains a KRAB (Krüppel-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 with
methyltransferase activity (93) and flanked by zinc fingers, and a tandem array of C2H2 zinc fingers.

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 zinc
fingers (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 (Krüppel-associated box) domain (20).
It also contains an SSXRD domain (131). In PRDM9, the zinc
finger domain is a tandem array of C2H2 zinc fingers. A PRDM9
zinc finger domain is under strong positive selection at residues
involved 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 for
male and female fertility in mice (93). Prdm9 is also involved
in hybrid sterility in mice. Genetic incompatibilities involving
Prdm9, X-linked, and autosomal loci lead to defects during mei-
otic prophase (19, 66, 154).
Linkage
disequilibrium
(LD)-based hotspot:
the detection of
crossover activity
based on LD analysis.
LD is a measure of
whether alleles at two
loci coexist in a
population in a
nonrandom fashion
and provides a value of
sex-averaged historical
crossover activity
hotspots come from mapping CO hotspots
through various methods. As in mice, these
hotspots appear to be determined by the bind-
ing specificity of PRDM9. The first evidence
for this came from the identification of a DNA
motif present at 40% of linkage disequilibrium
(LD)-based hotspots that shows similarity to
the predicted PRDM9 binding sequence (160)
as well as from correlations between Prdm9
genotypes and CO localization in pedigree
analysis (12). These findings were extended to
hotspot analysis in human populations with dif-
ferent Prdm9 alleles (95), association studies
that illustrated the role of PRDM9 in CO lo-
calization (74, 122), and high-resolution anal-
ysis of specific hotspots where both COs and
NCOs were analyzed (15, 16). Strikingly, all
18 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 in
hotspot activity (16). Some questions remain,
particularly because several hotspots show dif-
ferent activity in individuals that carry different
Prdm9 alleles but do not contain DNA motifs
with significant similarity to predicted PRDM9
binding sites. One current limit to interpreting
these observations is that current understand-
ing of the DNA-binding specificity of PRDM9
zinc 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 contains
approximately 23,000 recombination hotspots
that are 1–2 kb in size, are spaced approxi-
mately 50–100 kb apart, display variable inten-
sities, and are located in genic and intergenic
regions, with a bias for being distant from TSSs
(51, 159). Thus, qualitatively, human hotspots
show similar properties to those described in
mice. 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, almost
all of the information we have about hotspots
in humans is based on CO activity alone. As
is outlined in the section From Formation to
Repair of DNA Double-Strand Breaks: Out-
line and Main Players, meiotic DSB repair has
two major outcomes (CO and NCO), and the
CO map need not be a direct equivalent of a
DSB map. Indeed, it is important to note that
large variation (up to 40-fold) in CO:NCO ra-
tios between human hotspots has been reported
(98), and the factors that influence these ratios
are not well understood. Genome-wide geno-
typing or sequencing of single sperm cells has
been recently described in two studies, and this
novel approach creates new possibilities for the
monitoring of CO and NCO activities during
human spermatogenesis (138, 239).
An LD-based map of hotspots in chim-
panzees (9) showed a good conservation of CO
activity with humans at a broad scale (several
Mb 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
582 de Massy

GC–biased gene
conversion (gBGC):
a process whereby
meiotic recombination
leads to an increase in
GC content among
products
SNP:
single-nucleotide
polymorphism
finger array of the various Prdm9 alleles. How-
ever, overall CO activity profiles around genes
and CpG islands are similar in humans and
chimpanzees, suggesting a role for chromatin
organization in DSB activity. Unlike in hu-
mans, regions around predicted PRDM9 bind-
ing sites in chimpanzees do not show detectable
increases in recombination activity (9). This is
probably due to the limitation of predicting
PRDM9 binding sites in silico.
Crossover Hotspot Map in Canis
familiaris: SPO11 Loses Its
Guide in Canidae
Searches for PRDM9 conservation among
mammals led to the surprising finding that
PRDM9 is probably nonfunctional in Canis lu-
pus familiaris. The only copy of PRDM9 in the
dog genome appears to be a pseudogene, and
phylogenetic analysis suggests that PRDM9,
conserved in vertebrates, was lost in the canid
lineage (192). This was confirmed by further
phylogenetic studies indicating that PRDM9
inactivation occurred sometime between 7 and
49 Mya, predating dog domestication and canid
diversification (10, 156). Hotspot distribution
was determined by population diversity anal-
ysis, and approximately 4,000 hotspots were
mapped in regions of 18 kb in size or larger, the
precision of mapping being limited by low poly-
morphism density. These hotspots, which are
likely 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 within
genes, but show a strong enrichment for GC
content. Using pandas and cats as outgroups to
determine substitution patterns, these hotspot
regions were characterized as having a strong
substitution bias toward GC. This GC enrich-
ment, which is much stronger than in human
hotspots, might be due to GC-biased gene con-
version (gBGC) (65), and its extent indicates
that 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 Hotspots
in Arabidopsis thaliana
Evidence for highly localized recombination
activity 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 pollen
and of population diversity. Progeny analysis
first showed that small, several-kb-long regions
have a higher rate of recombination than the av-
erage of flanking sequences (64). In a study in
which meiotic recombination was detected by
high-throughput sequencing of the four prod-
ucts of a single meiosis, both COs and NCOs
were detected. COs were detected at the ex-
pected rate from the genetic map, but only one
to three NCOs per meiosis could be identified,
which is much lower than expected from the
counts of RAD51 or DMC1 foci (Table 1).
A significant fraction of NCOs may be un-
detectable in this analysis if gene conversion
tracts do not include single-nucleotide poly-
morphisms (SNPs), which depends on SNP
density and gene conversion tract length (137).
In contrast, by high-throughput sequencing of
F2 progeny from a cross with the same SNP
density as the previous study, a large number
of COs and also of NCOs could be detected
at 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, suggesting
that repeated sequences may interfere with the
analysis. The high number of NCO events
detected (from 265 to more than 3,000 per
meiosis, depending on the estimation method),
which may be consistent with the cytological
data, is however in contradiction with the other
study (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, with
a CO activity 200 times stronger than the
genome-wide average. These hotspots showed
a slight preference for location outside genes
(118) but determinants for their localization
are unknown. This tendency toward hotspot

localization in intergenic regions was con-
firmed in a large-scale population study that
also revealed a higher recombination rate
within transposable elements (99). Hotspots
have been identified in other plant species
by genotyping, specifically near the Hga1 and
Hga3 genes (200) in wheat, and within the a1-
sh2 region (253) and at the bz gene in maize
(63).
Crossover and Noncrossover Hotspots
in Drosophila melanogaster
The pioneering study of meiotic recombina-
tion events at the rosy locus of D. melanogaster
led to the proposal that initiation may take
place at multiple locations in the region stud-
ied (47). This conclusion may actually rep-
resent a general feature of the distribution
of meiotic recombination in D. melanogaster:
A genome-wide, high-resolution map of COs
and NCOs in D. melanogaster was generated
by progeny analysis using high-throughput se-
quencing (48). Overall, among more than 5,800
meioses, 32,511 COs, and 74,453s NCO were
identified. Interestingly, NCOs showed a much
more even distribution than did COs, and
NCOs were detected in regions with low or
no CO activity, such as telomeres, centromeres,
and the heterochromatic chromosome 4. This
result re-emphasizes that DSB activity can-
not be directly extrapolated from a CO map.
Among the events that could be mapped at
high resolution (+/− 500 bp, 5% of all events),
NCOs showed a slight tendency to be located
in genes (70% instead of the 60% expected
in the absence of bias) but were not close
to TSSs. In contrast, COs did not show any
bias 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 number
of heterogeneous DNA motifs were identified
as being enriched in hotspot regions, but the
significance of these motifs remains to be an-
alyzed. A study of CO distributions in a 2-Mb
region, among selected recombinant progeny,
also revealed large variation in CO activity
(209).
MEIOTIC RECOMBINATION AT
SPECIFIC GENOMIC REGIONS
One major challenge created by the pro-
grammed induction of DSB formation during
meiosis is the need to ensure the proper repair
of all DSBs, with a minimum of deleterious
events (deletions, duplications, and transloca-
tions) that compromise genome integrity. The
potential link between meiotic recombination
and loss of genome integrity, particularly those
events associated with human diseases, has been
reviewed (203). Recombination between re-
peated sequences can frequently lead to such
loss 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 sequences
either flank or are within centromeres, COs
within centromeric regions are extremely rare
or undetectable. We currently do not know
if this is due to a lack of DSBs or to a specific
channeling of DSB repair toward NCO or
intersister recombination. In S. pombe, pericen-
tromeric and centromeric chromatin is silenced
by RNA-interference (RNAi) pathways, and
several components of these pathways are
required for the repression of DSB formation
at these regions (68). Rec12 (Spo11) binding to
the centromeric core region of S. pombe chro-
mosomes was detected in one assay in which
Rec12 was cross-linked to chromatin in rad50S
strains, but not in other experiments per-
formed without cross-linking (54, 139). ChIP
experiments performed with cross-linking have
shown that S. cerevisiae Spo11 is transiently
enriched in a 20–30-kb region around this
organism’s 120-bp-long centromeres at the
beginning of meiotic S phase, but Spo11
then relocalizes to sites on chromosome arms
584 de Massy

Long terminal repeat
(LTR): repeated
regions located at both
ends of some
transposable elements
during prophase (123). Whether Spo11 has an
activity in this pericentromeric region remains
to be determined. DSBs are detected near
centromeres but at frequencies that are reduced
relative 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 localizes
at centromeres at the onset of prophase and
before DSB formation (166, 219). In zip1
mutants, levels of meiotic COs and NCOs are
increased in pericentromeric regions, whereas
DSB levels remain as they are in wild-type cells
(44). This finding suggests that Zip1 protein
may somehow direct DSBs that form near the
centromere toward intersister and away from
interhomolog recombination.
Telomeres
Analysis of recombination in telomeric regions
is inherently difficult because of the presence
of repeat sequences and the limited availability
of 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-wide
average) in sequences within 20 kb of telomeres
(23, 36, 180); the regions affected tend to be
gene-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, one
study reported increased DSBs, relative to the
genome as a whole, in the flanking regions 20–
100 kb from telomeres (44), although this result
was not observed in two other genome-wide
studies (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 subtelomeric
regions have also been observed during male
meiosis in several mammals (106, 121) as well
as in plants (80). These regional effects could
be related to properties of nuclear architecture,
chromosome organization, and/or chromoso-
mal positions within the nucleus that might be
influenced by interactions between telomeres
and the nuclear envelope.
Other Repeats
Ribosomal DNA (rDNA) genes are usually
found in a major repeated DNA cluster and
are composed of tandemly repeated genes with
a specific chromatin conformation and nuclear
organization. In S. cerevisiae, DSBs are almost
absent from the 1-Mb rDNA gene cluster (23),
and this repression depends on the histone
deacetylase Sir2 (153). Interestingly, the edges
of 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 mechanism
that involves the AAA+ ATPase Pch2 (231).
In addition, nontandem repeated sequences in
the yeast genome, such as retrotransposable el-
ements of the TY family and their long terminal
repeats (LTRs), have very low (but detectable)
DSB activity (180). This partial suppression is
associated with a closed chromatin configura-
tion, at least in the Ty element studied (14).
An unexpected result from high-resolution
mapping of human hotspots was the detec-
tion of hotspot activity within the THE1A and
B 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 the
CCTCCCTNNCCACsequencethatisabind-
ing site for the major human PRDM9 variant.
This feature is not a general property of repeat
DNA given that L1 elements are underrepre-
sented among hotspots (149, 161). Given the
postulated mechanism of action of PRDM9 in
hotspotspecificationthroughsequence-specific
DNA binding, hotspot activity in the THE1
repeat family is expected to be restricted to
individuals carrying Prdm9 alleles that recog-
nize the core motif mentioned above. Indeed,
hotspots maps derived from African popula-
tions, which contain different Prdm9 alleles, do
not show increased activity at THE1 elements,
and little evidence was found in these maps for

increased recombination activity in repeated el-
ements, except for a weak activity at the repeat
L1PA10 and L1PA13 from the L1 family (95).
HOTSPOT DYNAMICS
AND EVOLUTION
DSB repair involves the directional transfer
of genetic information from the noninitiating
chromatid to the initiating chromatid. This
transfer bias, which has been demonstrated
in fungi and in mammals, is thought to be
due, in part, to directionality of the mismatch
repair machinery that results in alleles that
are present on the initiating chromatid and
close to the DSB being converted to the other
parental genotype (190). This biased conver-
sion can occur in both NCO and CO path-
ways, as illustrated in Figure 1. Thus, alleles
that stimulate DSB activity in cis should grad-
ually be eliminated by this process, leading to
the loss of DSB activity. The continued pres-
ence of DSB activity, when, according to this
hypothesis, it should have been eliminated over
time, 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 included
in conversion tracts. However, in humans and
mice, because recombination is promoted by
sequence-specific binding of PRDM9, and be-
cause PRDM9 binding sites are predicted to
be close to initiation sites and thus to be fre-
quently converted, the paradox is predicted to
hold. In humans and mice, several sequence
changes near hotspot centers, located within
predicted PRDM9 binding sites, have been
shown to affect initiation activity. These SNPs
are included in conversion tracts and display
the expected transmission bias (15, 16, 87, 105).
Thus, PRDM9 high-affinity binding sites are
expected to decrease in frequency throughout
the genome during evolution. This prediction
is supported by comparison of chimpanzee and
human hotspots, which show an erosion of the
13-nucleotide motif related to the binding site
of the major PRDM9 variant in the human pop-
ulation (160).
A potential answer to the hotspot paradox
in mammals may involve changes in PRDM9
DNA-binding specificity. Prdm9 is among the
fastest evolving genes in the human genome
(177, 192, 227), and PRDM9 zinc finger
residues that are important for DNA sequence
bindingspecificityareunderconcertedandpos-
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 high
potential for recombination between repeats. A
very 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 array
by mitotic and meiotic recombination (103).
This high mutation rate predicts that changes
in hotspot usage may also occur rapidly, and
this may actually resolve the hotspot paradox
because the formation of mutations suppress-
ing hotspot activity in cis would be slower than
changes of hotspot usage due to Prdm9 instabil-
ity (103). However, it remains to be determined
how these dynamic properties impact hotspot
lifespan and recombination activity, and how
selection actually acts on Prdm9.
On the basis of calculations of changes in
GC content over evolutionary time, it was pro-
posed that hotspots in the dog genome may be
more stable than hotspots identified in humans.
Given the absence of Prdm9 in Canidae (see
above), it is possible that in dogs, the location
and activity of hotspots are not determined by
the local DNA sequence of sites where DSBs
and gene conversion occur. If properties such
as DNA accessibility are sufficient to determine
hotspot activity or if DNA motifs play a minor
role, as in S. cerevisiae, the impact of the dispar-
ity of gene conversion should be very weak or
absent at dog recombination hotspots. To eval-
uate these suggestions, it will be important to
have more direct information on hotspot sta-
bility in Canidae.
In other organisms, the NDRs where
hotspots occur may also have other roles,
586 de Massy

particularly in gene expression, and thus be
under selection. In this regard, a compar-
ison between S. cerevisiae meiotic recom-
bination maps and Saccharomyces paradoxus
LD-based recombination hotspots indicates
that hotspot activity is significantly conserved
among 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 meiotic
chromosomes 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 in
positive and negative regulation of DSB formation.
4. Sites of DSB formation are determined by several layers of control: (a) Accessibility for
Spo11. This accessibility depends on local properties of DSB sites, such as chromatin
structure and binding of transcription factors, and may also be influenced by chromosome
organization. In mammals, an alternative mechanism involving the DNA-binding protein
PRDM9 appears to promote the recruitment of Spo11. (b) DSB sites should also be
in sequences that associate with the chromosome axis before or at the time of DSB
formation. Several proteins mediate or stabilize these interactions, with two important
mediators being Spp1 in S. cerevisiae and Mde2 in S. pombe.
5. One consequence of the mechanisms involved in DSB formation is a highly punctuated
pattern of DSB localization. Whether this is the case in all organisms remains to be
determined. The role of DSBs in stabilizing homologous interactions in several organ-
isms probably requires DSB formation at multiple sites with a broad distribution along
chromosomes.
6. Hotspot locations appear to be highly dynamic in mammals, a consequence of the rapid
evolution of the DNA-binding domain of PRDM9. This indicates a great flexibility in the
localization of meiotic DSB events. The evolutionary constraints on hotspot localization
and the consequences of these dynamic changes on genome evolution remain to be
understood.
FUTURE ISSUES
1. The molecular machinery for DSB formation remains to be characterized, starting with
the enzymatic activity of Spo11, obviously a key point to clarify. The other proteins
involved may have regulatory or structural roles. Insight might thus be gained through
the identification of this regulation, possibly involving different protein modifications
and interactions with chromosome axis proteins.
2. How DSB formation is regulated in time and space still remains to be understood. Insight
into this process may be gained from understanding of the role of ATM and other DNA
damage-response kinases in regulating DSB formation.

3. The available high-resolution methods (single cell– or population-based) are extremely
useful, but ideally new strategies should be developed to follow the dynamics of DSB
formation and the interaction of DSB sites with the chromosome axis. Insight into the
organization of chromosome loops and the chromosome axis is essential for further
progress on the mechanisms and regulation of DSB formation and meiotic recombination
in general.
4. The consequences of hotspot localization for genome evolution remain unclear, both at
the molecular and evolutionary levels. What is the significance of DSBs being close to
or far from regions involved in gene expression?
5. It has been difficult to evaluate the impact of gene conversion events on genome dynamics
because 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 of
data regarding meiotic recombination events. The evolutionary consequences of having
stable or dynamic DSB sites remain to be understood.
6. Although the focus of this review has been on the molecular mechanisms and properties
that are required for normal recombination activity, mechanisms may exist that prevent
meiotic recombination from occurring in specific circumstances, e.g., in the prevention
of recombination between nonhomologous or partially homologous chromosomes. This
issue can be addressed from a mechanistic point of view but also from an evolutionary
point of view given that recombination barriers caused by genetic divergence may provide
a link between recombination and speciation.
DISCLOSURE STATEMENT
The author is not aware of any affiliations, memberships, funding, or financial holdings that might
be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
I thank Mathilde Grelon and Jerome Buard for critical reading of the manuscript and all members
of my laboratory for discussions about the various topics presented in this review. My group is
funded by grants from CNRS, ANR (ANR-09-BLAN-0269-01), and FRM.
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