Protein Determinants of Meiotic DNA Break Hot Spots
Kyle R. Fowler,1Susana Gutie ´rrez-Velasco,2Cristina Martı ´n-Castellanos,2,* and Gerald R. Smith1,*
1Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
2Instituto de Biologı ´a Funcional y Geno ´mica (Consejo Superior de Investigaciones Cientı ´ficas-Universidad de Salamanca),
C/ Zacarı ´as Gonza ´lez s/n, 37007 Salamanca, Spain
*Correspondence: email@example.com (C.M.-C.), firstname.lastname@example.org (G.R.S.)
Meiotic recombination, crucial for proper chromo-
some segregation and genome evolution, is initiated
yeasts and likely all sexually reproducing species. In
fission yeast, DSBs occur up to hundreds of times
more frequently at special sites, called hot spots,
than in other regions of the genome. What distin-
guishes hot spots from cold regions is an unsolved
problem, although transcription factors determine
some hot spots. We report the discovery that three
coiled-coil proteins—Rec25, Rec27, and Mug20—
bind essentially all hot spots with great specificity
even without DSB formation. These small proteins
are components of linear elements, are related to
synaptonemal complex proteins, and are essential
cate these hot spot determinants activate or stabilize
the DSB-forming protein Rec12 (Spo11 homolog)
rather than promote its binding to hot spots. We
propose a paradigm for hot spot determination and
crossover control by linear element proteins.
During meiosis, a defining feature of all sexually reproducing
species, homologous chromosomes segregate from each other
to convert diploid cells into haploid cells (eggs and sperm in
animals,ovulesandpollenin plants,orspores infungi).Homolog
segregation requires in most species a physical connection
between them, which imparts tension when homologs begin
to segregate properly to opposite poles at the first meiotic divi-
sion. The physical connection, a crossover, arises by homolo-
gous recombination, which also reassorts genetic differences
between homologs, thereby increasing the genetic diversity
important for evolution.
In the species most thoroughly studied at the molecular level,
the budding yeast Saccharomyces cerevisiae and the fission
yeast Schizosaccharomyces pombe, meiotic recombination is
initiated by DNA double-strand breaks (DSBs) formed by a topo-
isomerase II-like protein Spo11 (Rec12 in fission yeast) (Keeney,
2007). Because a Spo11 ortholog is found in all sexually repro-
ducing species examined, DSBs likely initiate meiotic recombi-
nation in all species; indeed, Spo11-deficient mutants of worms,
flies, mice, and plants are deficient in meiotic crossing-over.
During DSB formation, a tyrosine residue in the active site of
Spo11 becomes covalently linked to the DNA via a 50phospho-
diester bond. Unliketopoisomerase IIenzymes, however, Spo11
partner proteins for its action. Nine such partner proteins have
been identified in budding yeast, and six in fission yeast. Several
additional proteins, discussed below, strongly stimulate but are
not absolutely essential for DSB formation.
Meiotic DSBs are not uniformly distributed across the
genomes studied. Instead, there are preferred sites, called hot
spots, of DSB formation. What determines hot spots has been
a long-standing problem that is addressed here. In a few known
sequence bound by a transcription factor. At the HIS4 locus of
S. cerevisiae, Bas1, Bas2, and Rap1 factors bind closely spaced
sequences and increase DSB formation nearby (White et al.,
1993). Elimination of Bas1 decreases DSB formation at eight
other genomic sites but,curiously, alsoincreases DSB formation
at four others and has no significant effect at 58 other Bas1-
binding sites (Mieczkowski et al., 2006). The ade6-M26 single-
base-pair mutation of S. pombe creates a binding site for the
Atf1-Pcr1 transcription factor, which is essential for increased
gene conversion conferred by the M26 hot spot (Kon et al.,
1997). Elimination of Pcr1 reduces DSB formation at the M26
hot spot and at about a dozen selected sites with the DNA-
binding sequence (Steiner and Smith, 2005); however, only
a minority of DSB hot spots are likely bound by Atf1 (Cromie
et al., 2007; Eshaghi et al., 2010; unpublished data). Other tran-
scription factors also activate recombination hot spots in
S. pombe ade6 mutants containing their cognate binding
sequences (Steiner et al., 2011). Because there are hundreds
or thousands of DSB hot spots in S. pombe and S. cerevisiae,
respectively, no single transcription factor seems responsible
for most or all DSB hot spots. Collectively, transcription factors
may account for hot spots (Wahls and Davidson, 2010;
Pan et al., 2011), but too few data are available to allow a firm
conclusion. Thus, although a few hot spots clearly are deter-
mined at least in part by sites bound by transcription factors,
widespread protein determinants that bind to the hot spots
were until now unknown.
Overall chromatin structure also appears to strongly influence
hot spot activity. In S. cerevisiae, DSB hot spots often occur
in nucleosome-depleted regions (NDRs) (Pan et al., 2011). In
S. pombe, hot spots often contain an NDR over a small fraction
of the DSB region, but most NDRs are not near hot spots, indi-
cating that NDRs are poor predictors (de Castro et al., 2012).
Set1, a histone H3 Lys4 methyltransferase, is important for
most but not all DSB formation at the majority of hot spots in
Molecular Cell 49, 983–996, March 7, 2013 ª2013 Elsevier Inc. 983
S. cerevisiae, but set1D mutants have high spore viability (Borde
et al., 2009); thus, this chromatin feature is not required for
crossing-over. The Prdm9 H3 Lys4 methyltransferase is essen-
tial for recombination stimulation at hot spots in some mammals
but apparently not others (e.g., Baudat et al., 2010; Mun ˜oz-
Fuentes et al., 2011), and not all Prdm9-binding sites are hot
spots (Wang et al., 2012). The role of histone methylation in
DSB formation is currently unclear (Tischfield and Keeney,
2012). The failure of hot spots, including the well-defined M26
hot spot of S. pombe, to act when transplaced from their active
locus may reflect long-distance effects of chromatin structure
(Ponticelli and Smith, 1992). Furthermore, M26 hot spot activity
depends in part on histone modifications and chromatin remod-
eling factors (Hirota et al., 2008). Additional proteins that bind to
chromatin during meiosis to form the axial element precursors of
the synaptonemal complex (SC), such as S. cerevisiae Red1 and
Hop1, are also important for DSB formation (Keeney, 2007).
Meiotic cohesins are required in some regions but not others
for DSB formation in both S. pombe and S. cerevisiae (Ellermeier
and Smith, 2005; Kugou et al., 2009). In none of these cases,
however, are these chromatin-modifying factors known to
directly bind and activate hot spots with high specificity.
S. pombe lacks a full-fledged SC but has structures, called
linear elements (LinEs), whose temporal appearance and
morphology by electron microscopy of nuclear spreads are
similar to those of the axial element precursors of the SC of
S. cerevisiae (Loidl, 2006). LinEs may serve a role similar to that
proteins colocalize by light microscopy, and focus formation of
one dependsonthe others,implyingthatthey intimatelyinteract,
perhaps by forming a complex; indeed, Rec10 coimmunopreci-
pitates with the other three proteins, and in two-hybrid assays
Rec10 interacts with Rec25 and Rec15, a Rec12 partner protein
(Spirek et al., 2010; Miyoshi et al., 2012). rec10D mutants are
nearly as defective as rec12D mutants in the formation of re-
combinants (Ellermeier and Smith, 2005), but rec25D and
A pathway for S. pombe meiotic recombination
Figure 1. Pathway of Meiotic DSB Forma-
tion and Repair in S. pombe
About the time of replication, loading of meiotic
cohesin subunits Rec8 and Rec11 is followed by
loading of LinE proteins Rec10, Rec25, Rec27,
and Mug20and loadingor activation ofRec12 and
its six partner proteins. Rec12 makes DSBs,
becoming covalently linked to the DNA. Removal
of Rec12 allows repair of the DSB with the sister
chromatid or homolog. Repair with the homolog
can form a crossover, which allows proper
segregation of homologs at the first meiotic divi-
sion. See also Figure S5.
rec27D mutants retain significant, though
reduced, levels of recombination (Davis
et al., 2008), as do mutants lacking the
meiosis-specific Rec8 or Rec11 subunits
of sister chromatid cohesin and the non-
null rec10-109 mutant (DeVeaux and Smith, 1994; Ellermeier
and Smith, 2005). These phenotypes are understandable
because LinE focus-formation and binding to chromosomes
depend on meiotic cohesins, but cohesins form foci equally in
the absence or presence of LinE proteins (Molnar et al., 1995,
2003; Lorenz et al., 2004; Davis et al., 2008; Miyoshi et al.,
2012). These gene dependencies and physical interactions are
summarized in the pathway for DSB formation in S. pombe
shown in Figure 1: binding of Rec8 and Rec11 leads to assembly
of LinEs (Rec10, Rec25, and Rec27), which in turn leads to the
stabilization or activation of Rec12 and its partner proteins and
the formation of DSBs (Davis et al., 2008).
The phenotypes of mutants lacking cohesin subunits or LinE
components suggest that these structures are required for most
of the corresponding rec mutations, we have analyzed Rec12-
DNA covalent complexes, a measure of DSBs, genome-wide
proteins along the genome by immunoprecipitation of chromatin
crosslinked to each green fluorescent protein (GFP)-tagged
protein followed by microarray hybridization (ChIP-chip). Our
results show, unexpectedly, that Rec25, Rec27, and Mug20 are
enriched with exceptionally high specificity at DSB hot spots
and that Rec27 is required for formation of nearly all DSBs at
hot spots. Furthermore, hot spot DNA is bound by Rec27 even
without DSB formation, and mutants with altered Rec27 binding
have correspondingly altered hot spot DSB formation. Rec27
and Mug20 respectively show similarity to C. elegans SYP-2, an
SC component, and DDL-1, which interacts with SYP-2 (Colaia ´-
covo et al., 2003; Simonis et al., 2009). Thus, these proteins,
whose functions may be conserved for meiosis in other species,
are highly specific determinants of essentially all meiotic DSBs.
Linear Element Proteins Rec25, Rec27, and Mug20
Interdependently Colocalize in Meiotic Nuclei
Mug20 was identified in a Rec10 immunoprecipitate by mass
spectrometry (Spirek et al., 2010). This 17 kDa protein is induced
Protein Determinants of Meiotic DSB Hot Spots
984 Molecular Cell 49, 983–996, March 7, 2013 ª2013 Elsevier Inc.
early in meiosis, as are the 16–17 kDa proteins Rec25 and
Rec27 (Davis et al., 2008; Estreicher et al., 2012). We analyzed
byfluorescence microscopycellsbearingthe mug20-GFPfusion
gene and synchronously induced for meiosis in pat1-114 (Ts)
diploid strains. (The encoded fusion protein is nearly fully active
for recombination [Table S1 available online].) In intact cells,
Mug20-GFP behaved much like Rec25-GFP and Rec27-GFP:
all were visible from about 1.5 to 2 hr after meiotic induction,
when cells were replicating DNA, to about 4 hr, when cells were
beginning the first meiotic division; at 3.5 hr, when proteins and
DSBs were analyzed below, all three proteins are prominent in
most cells (Figures 2A, S1, S2, and S3; Davis et al., 2008).
Mug20-GFP was completely lost in the rec10D, rec25D, and
rec27D mutant cells (Figures 2A and S4A; unpublished data for
other induction periods). Conversely, in mug20D mutant cells
foci of Rec25-GFP and Rec27-GFP were completely lost, and
Rec10-GFP foci were less distinct (Figure 2B; unpublished data
for other induction periods). Like the LinE components Rec25
foci or elongated structures in rec8D mutants (Figure 2A); Rec8-
GFP, however, formed normal grainy nuclear structures in
mug20D cells (Figure S4B), as we previously showed for Rec8
in rec10D, rec25D, and rec27D mutants using nuclear spreading
(Davis et al., 2008). Also like the LinE component Rec10 (Lorenz
et al., 2004; Davis et al., 2008), Mug20-GFP formed abundant
structures in nuclear spreads of control and rec12D mutant cells
during meiotic prophase (Figure S4A and unpublished data).
Furthermore, Rec25-dtTomato and Mug20-GFP colocalized
during meiotic prophase in zygotic pat1+meiosis (Figure 2C); co-
localization was complete throughout pat1-114 prophase as well
The strikingly similar behavior of Mug20 and other LinE
components and the interdependence of the four proteins for
LinE component and confirm that LinE formation depends on
sister chromatid cohesins but not on DSB formation, as shown
in the pathway in Figure 1. Recently, Estreicher et al. (2012)
also showed that Mug20, in nuclear spreads rather than in intact
cells as used here, is a LinE-associated protein. We infer that the
dots in intact cells, whether fixed or not, reflect the in vivo struc-
tures and that the proteins may become reorganized during
In spite of clear similarities, there are differences in the
behavior of the four LinE components. Rec10-GFP remained
primarily in the nucleus in the absence of Mug20, whereas
Rec25-GFP, Rec27-GFP, and Mug20-GFP were evenly distrib-
utedthroughout the cell in the absence of any other LinE compo-
has a predicted nuclear localization sequence but the other
proteins do not (Lorenz et al., 2004 and unpublished data).
Rec10-GFP, Rec25-GFP, and Rec27-GFP foci are sharper than
those of Mug20-GFP, which appeared to make some thin lines
as well as fuzzy dots (Figures 2A, S1, S2, and S3) (Davis et al.,
2008). Although DSB formation and recombination are essen-
tially eliminated in rec10D mutants (Ellermeier and Smith, 2005)
nation in rec25D and rec27D mutants (Davis et al., 2008) as well
as in mug20D mutants (Table S1; Estreicher et al., 2012). We
discuss the implications of these observations later.
Conservation of Coiled-Coil Domain Proteins Rec27 and
Mug20 among Species
Three LinE proteins (Rec25, Rec27, and Mug20) are rather small
proteins with predicted coiled-coil domains also found in SC
proteins and some DNA-binding transcription factors, although
LinE and SC proteins lack an obvious DNA-binding motif. We
compared the amino acid sequences of these three LinE
proteins encoded by four Schizosaccharomyces species with
the sequences of the small (?25 kDa) SC proteins SYP-2 and
SYP-3 of four Caenorhabditis species and found remarkably
conserved similarity among the Rec27 and SYP-2 proteins (Fig-
ure S5). The similarity encompasses the predicted coiled-coil
domain as well as a region toward the N terminus. Like Rec27,
localization of SYP-2 on meiotic chromosomes strongly
depends on Rec8 but not Spo11 (Rec12) (Colaia ´covo et al.,
2003). We also found similarity among Schizosaccharomyces
Mug20 and Caenorhabditis DDL-1 proteins; in a two-hybrid
assay DDL-1 interacts with SYP-2 (Simonis et al., 2009), sug-
gesting that DDL-1 is associated with the SC. Although LinEs
have been considered to be only distantly related to SCs (Loidl,
2006), these similarities suggest a more highly conserved func-
tion common to the two structures than previously realized.
Rec25, Rec27, and Mug20 Colocalize at DSB Hot Spots
The preceding microscopic analyses suggested that Rec25,
Rec27, and Mug20 bind chromosomes at the same or closely
linked sites. To determine their localization at high resolution,
we analyzed by ChIP-chip the localization of the GFP-tagged
proteins described above. We found that all three proteins are
highly enriched at certain sites across the genome. Figure 3A
shows a representative 1 Mb interval of the 12.5 Mb
genome; graphical representation of the data in Figures 3
and 6 for the whole genome is on the lab website (http://labs.
Supplemental_Figures_2013.pdf). In this 1 Mb interval there are
25 DSB hot spots, taken as sites at which >0.3% of the DNA is
broken, as determined by the nearly linear relation, for 25 hot
spots, between Rec12-DNA covalent linkages and DSBs as-
sayed directly by Southern blots (Cromie et al., 2007); this DSB
level is at the limit of detection by Southern blots. At some hot
spots in the genome, these linkages are >250 times the genome
median, as in previous analyses (Cromie et al., 2007; Hyppa
et al., 2008). Linkages were determined in rad50S mutants, in
which DSBs are made with the same distribution as that in
rad50+but are not repaired and hence accumulate, allowing
sensitive measures of DSBs (Hyppa et al., 2008). Between these
hot spots, Rec12-DNA linkages do not rise significantly above
the genome median, defining cold regions.
The distribution of each of the three LinE proteins (Rec25,
Rec27, and Mug20) closely parallels that of DSBs. For example,
Rec25 was enriched more than two times the genome median at
23 of the 25 DSB hot spots in the 1 Mb interval shown in Fig-
ure 3A, whereas between the hot spots Rec25 was found at
essentially the genome-median level. Enrichments up to 80
Protein Determinants of Meiotic DSB Hot Spots
Molecular Cell 49, 983–996, March 7, 2013 ª2013 Elsevier Inc. 985
Figure 2. Mug20 Is a LinE Component Interacting with Rec25, Rec27, and Rec10
(A and B) Cells with the indicated GFP fusion protein and rec gene were synchronously induced for meiosis, fixed 3 hr later, stained with DAPI, and examined by
fluorescence microscopy. Eachset of three images shows theGFP protein (green; left), DAPI-stained DNA(blue;middle),and merge (right). Mug20 formsnuclear
foci that depend on LinE components Rec10, Rec25, and Rec27 but only partially on the sister chromatid cohesin Rec8 (A). Mug20 is required for nuclear focus
formation of Rec25 and Rec27 and, partially, of Rec10 (B).
zygotic cells. See also Figures S1, S2, S3, and S4.
Protein Determinants of Meiotic DSB Hot Spots
986 Molecular Cell 49, 983–996, March 7, 2013 ª2013 Elsevier Inc.
times the genome median were found at some hot spots (lab
website). Rec27 and Mug20 showed similar enrichments of up
to 24 and 28 times the genome median, respectively (Figures
3A). The binding profiles of all three proteins closely parallel
that of DSBs, determined either by ChIP-chip analysis of
Rec12-DNA linkages or by high-resolution Southern blots
(Figures 3A and S6). We estimate from these data that the reso-
lution (maximal genome distance by which two peaks could be
offset but appear to be coincident) is <1 kb. Most hot spots,
such as the well-studied mbs1 hot spot, are clusters of closely
spaced sites of variable breakage; these DSB regions are up
to 7 kb wide and on average are ?45 kb apart (Figure S6; Cromie
Figure 3. Rec25, Rec27, and Mug20 Bind DNA at Meiotic DSB Hot Spots with High Preference, but Rec8, Rec10, Rec11, and Rec12 Bind with
Little or No Preference
DNA covalently linked to Rec12-FLAG (signifying DSBs; harvested at 5 hr, when DSBs are maximal) or DNA crosslinked to the indicated GFP fusion protein
(harvested at 3.5 hr, when foci are prominent) was analyzed by microarray hybridization. Data are median normalized, smoothed using an 11-probe window, and
plotted with an offset for legibility. ‘‘Input’’ is whole cell extract. Complete genome data are on the lab website.
Rec10 (left axis) binds nearly uniformly except for modest preference at strong hot spots. Black circles beneath the traces indicate wild-type hot spots (see
Supplemental Experimental Procedures for peak-calling criteria). See also Figure S6.
(B)Sistercohesin subunitsRec8and Rec11 (left axis; offsets of 1and 0,respectively) bindnearly uniformly, as does theinactiveRec12-213(Y98F)mutant protein
(left axis; offset of 2).
(C) DSBs are nearly eliminated in rec10D and are significantly reduced at most hot spots in rec27D and rec11D null mutants and the rec10-109 missense mutant
(left axis; offsets of 0, 5, 10, and 15, respectively).
(D) LinE protein Rec27 preferentially marks the DSB hot spots remaining in rec8D. See also Figure S7.
Protein Determinants of Meiotic DSB Hot Spots
Molecular Cell 49, 983–996, March 7, 2013 ª2013 Elsevier Inc. 987
To quantify the correlations, we compared the enrichments of
each of these proteins to those of the Rec12-DNA linkages at
each of the ?44,000 genome positions (probes) represented
on the microarray. Scatterplots showed that Rec25, Rec27,
and Mug20 are highly enriched at DSB hot spots (Figure 4A,
lated the Pearson correlation coefficient r. Without smoothing
of the data (i.e., analysis of all individual probes), r = 0.76, 0.75,
and 0.78 for Rec25, Rec27, and Mug20, respectively, versus
Rec12-DNA linkages (DSBs). These values rise only slightly to
0.80, 0.79, and 0.85, when smoothed over an 11-probe (?3 kb)
window (Table S2), an indication of the high precision and reso-
lution of these data. For comparison, we note that r for two inde-
pendent Rec27 protein distributions is 0.77 (Figure 4C), which
we take as the upper bound for identity of genome-wide features
determined on microarrays. Thus, these data show that Rec25,
Rec27, and Mug20 are enriched nearly exclusively, and excep-
tionally highly, at DSB hot spots. These proteins may also be
Figure 4. Rec25, Rec27, and Mug20 Binding Is Highly Correlated with Genome-wide DSB Frequencies, but Rec8, Rec11, and Rec12 Binding
(A and B) Scatterplots of the genome median-normalized data for the two parameters indicated on the axes. All data points (?44,000; in black) are plotted on
a log10scale (IP/input), but most are obscured by their high density. Red data are points within DSB hot spots. Pearson correlation coefficients (r) are for
(D) Scatterplots and r from two rec8D inductions, one of which was done concurrently with rec27D.
(E) Scatterplot and r, as above, for Rec12-213 (Y98F) and Rec12+proteins (lab website). The Rec12+data reflect both self-linkage (DSBs) and crosslinking
(binding), but a positive correlation is still observed.
Protein Determinants of Meiotic DSB Hot Spots
988 Molecular Cell 49, 983–996, March 7, 2013 ª2013 Elsevier Inc.
present in cold regions, but if so their frequency is, conserva-
tively, <10% of that at strong hot spots.
The correlation between protein binding and DSBs was also
dramatic when we analyzed hot spots individually. For this anal-
ysis, we integrated the values for DSB frequency and for protein
binding across each of the 288 hot spots in the genome (those
with >0.3% DSBs, as defined above). The data show that the
DSB frequency is a linear function of the relative amount of
protein bound (Figure 5); r =0.79, 0.83, and 0.88 for DSBs versus
Rec25, Rec27, and Mug20, respectively. Thus, binding of these
proteins determines not only the position of DSB hot spots, but
also accounts for the majority of the variation in breakage: the
coefficient of determination R2is 0.62–0.78. Similarly, in
rec8D, in which Rec27 binding is altered (Figure 3D), the few
hot spots that remain show an exceptionally strong correlation
(r = 0.95) between the amount of breakage and Rec27 bound
A corollary of these observations is that these three proteins
should be highly colocalized on the genome, as suggested by
the microscopy data (Figures 2, S1, S2, and S3; Davis et al.,
2008). The data in Figure 3A show that this is the case: Rec25,
Rec27, and Mug20 are enriched at only a limited number of sites
Scatterplots of these data confirm this conclusion. For example,
there is a high, linear correlation between the abundance of
Rec25 and Rec27, of Rec25 and Mug20, and of Rec27 and
Mug20 at each probe (Figure 4B). Thus, these data confirm the
colocalization implied by microscopy and show that these three
LinE proteins are strongly enriched exclusively at the same sites:
hot spots of DSB formation.
Linear Element Proteins and Cohesins Are Required for
DSB Formation at Most Hot Spots
Rec25 and Rec27 are enriched specifically at DSB hot spots
(Figures 3, 4, and 5) and are required for DSB formation at the
few loci previously tested by Southern blot hybridizations
(Martı ´n-Castellanos et al., 2005). To determine the extent of this
requirement, we determined the genome-wide DSB distribu-
at more than 80% of the hot spots in the rec27D mutant (Fig-
ure 3C). Low-level DSBs were seen at some of the strongest hot
spots, but even these DSBs were reduced or nearly eliminated.
Similar DSB patterns were seen in the absence of the meiosis-
specific cohesin subunits Rec8 or Rec11. DSBs at most of the
hot spots were nearly eliminated, and residual levels were seen
at the same hot spots at which DSBs remained in rec27D
(Figures 3C and 3D). There is a striking correlation between
Table S2). Thus, DSBs at most hot spots depend on both Rec8
and Rec27, in accord with Rec8 being required for binding of
Rec27 to most hot spots and the requirement of meiotic cohe-
sins for LinE focus-formation and localization on DNA (Molnar
et al., 1995, 2003; Lorenz et al., 2004; Davis et al., 2008; Miyoshi
et al., 2012).
A distinctly different pattern was seen in the rec10D mutant:
DSBs were completely eliminated except for an almost invisible
amount at an exceptionally strong hot spot near 4.0 Mb on the
right end of chromosome 2 (Figure 3C; see also lab website).
Southern blot analysis shows that the very low-level DSBs at
this site are meiosis specific (Ellermeier and Smith, 2005).
Thus, our ChIP-chip analysis has the power to detect even tiny
Figure 5. Correlations between DSB Frequencies and Protein Abundances at DSB Hot Spots Are Especially Strong
Scatter plots and r for DSBs (integral above or below median of Rec12-DNA covalent linkages across hot spots) and the indicated protein similarly integrated.
Points above the line indicate protein enrichment at that hot spot; points below imply depletion. Data are in arbitrary units on linear axes.
Protein Determinants of Meiotic DSB Hot Spots
Molecular Cell 49, 983–996, March 7, 2013 ª2013 Elsevier Inc. 989
amounts of DSBs. With this one exception, the DSB pattern in
rec10D was indistinguishable from that in rec12D, which lacks
the protein with the active site for DSB formation (Young et al.,
2002). In contrast, significant levels of DSBs remained in the
initial rec10 isolate, rec10-109, which harbors two closely
spaced missense mutations and retains significant region-
specific recombination (DeVeaux and Smith, 1994; Ellermeier
and Smith, 2005); the DSB pattern closely resembles that in
rec8D, rec11D, and rec27D mutants (Figures 3C and 3D). In
the Discussion, we propose an explanation for the DSB patterns
seen in these mutants.
Rec27 Binds Sites Poised To Be DSB Hot Spots Even in
the Absence of DSB Formation
Given the hot spot-enriched binding shown above and that LinE
components are required to form DSBs (Ellermeier and Smith,
2005; Martı ´n-Castellanos et al., 2005), we would expect these
proteins to be present before DSB formation and, thus, even in
the absence of DSB formation. To test this hypothesis, we deter-
mined the genome-wide distribution of Rec27 in the absence of
Rec12. We found that the distributions were practically identical
(Figures 4C and 6A; Table S2). The simplest interpretation of
these data and the genome-wide requirement for Rec27 for
most DSB formation is that Rec27 localizes to sites poised to
be DSB hot spots before DSBs are formed and recruits one or
more DSB-forming proteins to their sites of action or activates
them after they bind, or both.
The high correlation of LinE protein binding with both hot spot
position and hot spot intensity predicts that altering the LinE-
binding profile should alter the DSB landscape. Rec8 is required
forproper LinE formation (Molnaretal.,1995;Lorenz etal.,2004;
Davis et al., 2008), and a rec8D mutant has coordinately altered
DSB and Rec27-binding profiles (Figure 3D). Similarly, insertion
of exogenous DNA (the bacterial kan drug-resistance determi-
nant) at the rec8+locus creates both a Rec27-binding site and
a DSB hot spot (Figure S7); similar results were found with other
1000 1200 1400
Chromosome 1 (kb)
Rec27 IP / Input
WT (Exp. 2)
WT (Exp. 1)
700 710720 730740 750 760 770
Chromosome 1 (kb)
Rec12 IP / Input
Figure 6. Rec27 Binds to DSB Hot Spots in the Absence of DSB-formation by Rec12, which Binds to DSB Hot Spots with Only Modest
(A) Rec27 binding in rec12+(Figure 3, experiment 2, offset of 1) or in rec12D (offset of 2; analyzed concurrently with rec12+in experiment 2). r = 0.77 for single
probes and 0.81 for 11-probe smoothing (Figure 4C; Table S2). Complete genome data are on the lab website.
(B)Binding ofinactiveRec12-213(Y98F)asinFigure3islargelyindependentofRec27(Figure S8).Binding ishigherinprotein-coding genes(red andblue barsfor
upper-and lower-strand coding) and lower betweengenes. Inset: 4003geneswerealigned attheirtranscriptionstartsites (TSS) and transcriptionend sites(TES)
(Lantermann et al., 2010). Rec12-Y98F binding (red line) is higher in genes (black rectangle with arrow) than between genes, whereas mean DSBs (Rec12-DNA
covalent linkages; black line) is higher between genes than in genes. Note recombinant frequency in ura1 (7 kb blue bar near 740 kb) is 19 times lower than
genome average (Supplemental Information), although Rec12-Y98F binding is about twice the genome median. See also Figure S9.
Protein Determinants of Meiotic DSB Hot Spots
990 Molecular Cell 49, 983–996, March 7, 2013 ª2013 Elsevier Inc.
insertions, such as FLAG (unpublished data). The generation of
DSB hot spots at manipulated loci has previously been observed
in S. pombe and S. cerevisiae, but the mechanism remains unre-
solved (e.g., Ponticelli and Smith, 1992; Borde et al., 1999; de
Castro et al., 2012). Thus, sites poised for DSB formation, even
unusual de novo sites, are predetermined by Rec27, indicating
a mechanistic relationship between Rec27 binding and DSB
Rec12 Binds DNA with Only Modest Preference for DSB
The strong preference for DSB formation at hot spots could
erential activation there. To distinguish these possibilities, we
determined the genome-wide binding profile of Rec12 with
phenylalanine in place of the active-site tyrosine. This protein,
from the rec12-213 (Y98F) mutant, lacks a single oxygen
atom necessary for wild-type Rec12 DSB formation and is as
recombination deficient as rec12D (Cervantes et al., 2000).
Using this mutant protein eliminates self-linkage, which could
be exceptionally strong at DSB hot spots and thereby obscure
the true Rec12-binding profile. Rec12-213 (Y98F) binding
has only low-level peaks above the genome median across
most of the genome, although at exceptionally strong DSB hot
spots it is clearly more abundant than the genome median
(Figures 3B and 6B). A similar pattern was seen for DSB-profi-
cient Rec12 crosslinked with formaldehyde (Figures 4E and
S8; Ludin et al., 2008), indicating that Rec12 and Rec12-213
(Y98F) bind similarly.
The pattern of binding in the low-level regions, with multiple
adjacent probes significantly above the genome median, implies
that the low-level peaks in meiosis are not background ‘‘noise.’’
The distributions about these peaks are nearly identical for
Rec12 and Rec12-213 (Y98F) (Figure S8A), again indicating
that these proteins bind the same. Furthermore, Rec12-213
(Y98F) binds significantly above the genome median in genes
but less than the genome median between genes, whereas
differentials between genes and intergenic regions, both for
binding and DSB formation, increase with increasing abundance
of meiotic transcripts (Figure S9), suggesting that transcription
can promote Rec12 binding and DSB formation but in distinctly
separate regions. As expected, r for DSBs versus Rec12-213
(Y98F) binding is much lower than r for DSBs versus Rec27
binding, for example (Figures 4A and 4C). If only individual hot
spots are considered, however, r for DSBs versus Rec12-213
(Y98F) binding is 0.85, about the same as r for DSBs versus
Rec25, Rec27, or Mug20 (Figure 5); with hot spots excluded it
is ?0.05. Thus, although Rec12 binds to hot spots in proportion
to the amount of DSBs that will be formed, it also binds outside
hot spots but nearly at random with respect to the amount of
DSBs that will be formed. We account for this pattern in the
Rec12 Binds to Some DNA Sites Independent of Rec27
without Forming DSBs
We noted ?20 loci at which both Rec12 and Rec12-213 (Y98F)
bind significantly above the genome median, yet at which few
if any DSBs are formed. Two such sites, denoted C and D in
Figure S8B, are near two prominent DSB hot spots, denoted A
and B. Although Rec12-213 (Y98F) (with formaldehyde cross-
linking) is nearly equivalent at all four sites, DSBs (Rec12
self-linkages, without formaldehyde) are much more prominent
at A and B than at C and D. These data show directly that
Rec12 can bind without making DSBs and indicate that
Rec12 is activated at some sites (hot spots) by another factor.
One of these factors appears to be Rec27, because Rec27 is
abundant at sites A and B but not at C and D (lab website).
Furthermore, in the absence of Rec27, binding of Rec12 is abun-
dant at all four sites even though DSBs (self-linkage) are barely
detectable at these sites in rec27D (Figure S8B). Thus, Rec12
can bind without Rec27, but makes DSBs at most hot spots
only in its presence.
Meiotic Cohesin Subunits Rec8 and Rec11 and Linear
Element Protein Rec10AreNearly UniformlyDistributed
Across the Genome
Microscopic analyses show that most but not all LinE formation
requires Rec8 and Rec11 (Figure 2A; Molnar et al., 1995, 2003;
Lorenz et al., 2004; Davis et al., 2008). To determine if the hot
spot-specific binding of Rec25, Rec27, and Mug20 reflects
hot spot-specific binding of Rec8 and Rec11, we determined
the genome-wide distributions by ChIP-chip of Rec8-GFP and
Rec11-GFP, which are nearly fully active for DSB formation
and recombination (Table S1). Unexpectedly, these two
proteins were distributed nearly uniformly across the genome,
with no preferential enrichment or depletion at hot spots
(Figures 3B). Scatterplots confirm this impression (Figures 4A
and 4C). Considering hot spots individually, at roughly half of
the hot spots each protein is below the genome median, as ex-
pected for uniform binding with some variation; in sharp
contrast, the LinE proteins Rec25, Rec27, and Mug20 are en-
riched above the median at R94% of the hot spots (Figure 5).
These data suggest no significant correlation between Rec8
and Rec11 binding and DSB formation, a marked difference
from the negative correlation of DSBs and Rec8 binding in
S. cerevisiae (see Discussion) (Blat et al., 2002; Glynn et al.,
2004; Panizza et al., 2011). (Ding et al.  reported some-
what greater excursions in Rec8 binding density across the
part of the genome they assayed; this difference may reflect
the Rec8-HA tag or the use of haploids instead of diploids, as
We were further surprised by the distribution of Rec10, which
by fluorescence microscopy appears to colocalize with Rec25,
Rec27, and Mug20 (Davis et al., 2008; Estreicher et al., 2012).
Rec10-GFP binds nearly uniformly across the genome, but
with modest enrichment at many hot spots (Figure 3A). The
enrichment at hot spots was generally less than three times the
genome median, and the highest enrichment was 6-fold at an
exceptionally strong hot spot on the right end of chromosome
1. r for Rec10 versus DSBs is 0.65, slightly lower than that for
Rec27 (0.77) (Figure 4C). Considering only enrichment at hot
spots, r = 0.80 for Rec10 (Figure 5), and with hot spots excluded,
r = 0.35. Thus, DSB frequency and Rec10 abundance are more
highly correlated at DSB hot spots than in DSB-cold regions,
Protein Determinants of Meiotic DSB Hot Spots
Molecular Cell 49, 983–996, March 7, 2013 ª2013 Elsevier Inc. 991
It has long been recognized that meiotic recombination does not
occur at uniform frequency across the genome; rather, there are
hot spots of recombination—sites at which recombination
occurs at higher-than-average frequency—and intervening
cold regions (Keeney, 2007). But what determines hot spots
has been largely elusive except for a few particular sites acti-
vated by certain transcription factors and a more widespread
effect of chromatin structure (see Introduction). Here, we identify
three coiled-coil proteins, Rec25, Rec27, and Mug20, likely
acting asa complex, that bind to and, at least for Rec27, activate
nearly all DSB hot spots across the genome of the fission yeast
S. pombe. These proteins are components of linear elements
and are related to the SC proteins of other species (Loidl,
2006; Figure S5).This feature provides the basisfor anadditional
level of control, discussed below, for formation of crossovers,
the crucial connection between homologs that allows their
successful segregation in meiosis.
Rec25, Rec27, and Mug20 Bind Hot Spots with High
Specificity and Are Hot Spot Determinants
Microarray-based assays for binding of these three proteins
to DNA show directly that they are enriched with unprecedented
specificity at DSB hot spots, with an enrichment up to 80 times
the genome median (Figures 3A). Quantitative analysis shows
a linear relation between the frequency of DSB formation at a
hot spot and the frequency of protein binding at that hot
spot (Figure 5). Elimination of Rec25 or Rec27 protein strongly
reduces or eliminates DSB formation at hot spots (Figure 3C;
Martı ´n-Castellanos et al., 2005); to our knowledge, Mug20
has not been similarly tested. Thus, these proteins determine
both the position and the activity of nearly all hot spots across
the genome and can be considered essential components of
meiotic DSB hot spots. As predicted by this conclusion,
when exogenous DNA was inserted into the chromosome, it
created both a hot spot for Rec27 binding and a hot spot for
DSB formation (Figure S7). Furthermore, deleting rec8 coordi-
nately reduces DSB formation and Rec27 binding, leaving a
DSB landscape that mirrors the residual Rec27-binding profile
(Figures 3D and 5).
Previous reports have shown that certain transcription factors
thoroughly, Bas1 of S. cerevisiae, activates only a few of the
sequence for Atf1-Pcr1 is a poor predictor of DSB hot spots in
S. pombe (Steiner and Smith, 2005; Mieczkowski et al., 2006).
Other factors, including chromatin remodeling and histone
modifications, have more widespread effects (Hirota et al.,
2008; Borde et al., 2009; de Castro et al., 2012; Pan et al.,
2011), but it is not clear that these modifications act directly
(as opposed to altering replication or gene expression and
thereby having indirect effects on recombination), nor is it clear
that they are hot spot specific. Indeed, most such factors are
poor predictors of hot spots (Tischfield and Keeney, 2012).
Rec25, Rec27, or Mug20 detectably bind at 86% of all DSB
hot spots (97% of the hottest two-thirds of sites, or about 200
hot spots), and they are enriched nearly exclusively at hot spots,
making their binding the best predictor for hot spot position
in any species reported to date.
Meiosis-Specific Cohesin Subunits Rec8 and Rec11 and
Linear Element Protein Rec10 Bind Chromosomes with
Little Site Specificity
Meiotic cohesins are required for LinE formation (Figure 2; Mol-
nar et al., 1995, 2003; Lorenz et al., 2004; Davis et al., 2008;
Estreicher et al., 2012), and Rec8 and Rec11 make discrete
foci in chromosome spreads or live cells (e.g., Ding et al.,
across the genome (Figures 3B, 4A, and 4C). We suppose that
this uniformity reflects a limited amount of these proteins (to
account for their punctate foci in individual cells) that binds
with nearly equal probability at any point along the DNA (to
If so, on an individual chromosome a limited number of nearly
random sites may be bound by these meiosis-specific cohesins.
Rec10, the protein that defined linear elements seen by light
microscopy (Lorenz et al., 2004), also binds along the chromo-
somes nearly uniformly, although it binds somewhat more
frequently to DSB hot spots (Figure 3A). Miyoshi et al. (2012)
recently reported a similar profile during haploid meiosis:
Rec10-FLAG is modestly enriched at hot spots with more
uniform binding elsewhere. The relative magnitude of Rec10’s
binding to hot spots is not, however, as great as that of Rec25,
Rec27, or Mug20: the maximal enrichment of Rec10 at a hot
spot is six times the genome median, whereas the maximal
enrichments for Rec25, Rec27, and Mug20 are 80, 24, and 28
times the genome median, respectively (Figures 3A). The abso-
lute amount of Rec10 at a hot spot may be as high as that of
Rec25, for example, but if so then the level of Rec10 between
We infer that Rec10, like Rec8 and Rec11, binds nearly uniformly
across the genome but, unlike Rec8 and Rec11, with additional
enrichment at hot spots.
Rec10, Rec25, Rec27, and Mug20 Stabilize or Activate
Rec12 to Make DSBs, Rather Than Recruiting Rec12 to
DSB Hot Spots
The nearly uniform binding of Rec12, as the DSB-inactive Y98F
mutant, along chromosomes strongly contrasts with the much
higher specificity of DSB formation at hot spots (Figures 3B, 6,
and S8). In addition, Rec12 binds to many points along the chro-
mosome where it makes few if any DSBs (Figure S8). We
propose that Rec12 is stabilized or activated to a high level by
Rec25, Rec27, and Mug20, specifically at hot spots, and is acti-
vated at a low level by Rec10 alone, to make low-level DSBs
between hot spots (i.e., in DSB-cold regions). Because Rec10
is also required for Rec25, Rec27, and Mug20 focus-formation
(Figures 2A and S4; Davis et al., 2008) and Rec27, and perhaps
(Figures 3C; Martı ´n-Castellanos et al., 2005), this view predicts,
as observed, that Rec10 is essential for virtually all DSBs across
the genome (Figures 3C; Ellermeier and Smith, 2005).
Protein Determinants of Meiotic DSB Hot Spots
992 Molecular Cell 49, 983–996, March 7, 2013 ª2013 Elsevier Inc.
Our conclusion that Rec12 binds to both DSB-cold regions
and DSB hot spots but is activated at DSB hot spots by
Rec25, Rec27, and Mug20 is supported by the distinct pattern
of global Rec12 binding and DSB formation along the chromo-
some. In DSB-cold regions, low-level DSBs are more frequent
between genes, but Rec12 binding is more frequent within
genes (Figure 6B, inset). This preferential loading in genes
correlates with transcriptional activity and is strongly influenced
by the transcript start and stop sites (Figures 6B and S9).
Furthermore, in the ura1 gene recombination is about 20 times
lower than the genome mean, but Rec12 binding is about twice
the genome median (Supplemental Information). This inverse
relation is most readily explained by a requirement for Rec12
to be activated after it has bound DNA (i.e., loading is not suffi-
cient for breakage). At DSB hot spots, this activation depends
on Rec25, Rec27, and Mug20: the level of DSBs is proportional
to the amount of each of these three proteins bound (Figure 5).
DSB hot spots thus depend on the strong localization of
Rec25, Rec27, and Mug20 to hot spots. Our proposal also
explains the observation that only ?10% of the total Rec12 is
covalently linked to DNA (Milman et al., 2009). The majority
of bound Rec12 may have another role, such as chromosome
segregation at the second meiotic division (Sharif et al., 2002).
In S. cerevisiae and mice, there also appears to be a large
excess of the Rec12 ortholog, Spo11, which may play a role
independent of DSB formation (Keeney, 2007; Bellani et al.,
Rec12 may be activated for DSB formation by the binding of
one or more of its partner proteins, dependent on one or more
of the LinE proteins. Formation of nuclear foci by two Rec12
partner proteins, Rec7 and Rec24, depends on Rec10 (Lorenz
et al., 2006; Bonfils et al., 2011). One of these partner proteins,
suchasRec15, whichinteracts with Rec10 in atwo-hybrid assay
(Miyoshi et al., 2012), may be rate limiting for DSB formation and
more abundant at hot spots than in DSB-cold regions. Rec10
may be the crucial link between the ‘‘early’’ proteins (cohesins
and other LinE proteins) and the ‘‘late’’ proteins (Rec12 and its
partners) for DSB formation, with additional activation at hot
spots by Rec25, Rec27, and Mug20 (Figure 1).
Localization of Rec25, Rec27, and Mug20 to DSB Hot
Unlike the protein determinants, the DNA determinants of most
DSB hot spots remain unclear. Sequence comparisons, such
as that by MEME (http://www.meme.sdsc.edu), do not reveal
an obvious consensus sequence for hot spots, although poly-
purine stretches on one strand have a limited correlation with
hot spots (Cromie et al., 2007). Noncoding RNAs (ncRNAs) are
correlated with hot spots (Wahls et al., 2008), but this correlation
may simply reflect the higher-than-average density of ncRNA
genes in large intergenic regions and not be directly causative.
Because Rec10 binds to hot spots with modest preference
(Figures 3A, 4, and 5) and because formation of nuclear foci by
Rec25, Rec27, and Mug20 depends on Rec10 (Figures 2A and
S4A; Davis et al., 2008; Estreicher et al., 2012), we infer that
this protein complex has the intrinsic ability to bind hot spots;
theircoiled-coil structure suggeststheymayactlikecertain tran-
scription factors with extensive coiled-coil domains. The hot
spot specificity may reside within one of these proteins but be
effective only when in the putative complex.
This proposal is concordant with the chromosome interval-
dependent reduction of DSB formation and recombination in
rec25D, rec27D, mug20D, and rec10-109 mutants. In the
mutants tested in this set, DSBs are strongly reduced at most
hot spots (Figures 3C; Davis et al., 2008), and recombinant
frequencies are reduced in some intervals by factors of >100
but in other intervals by factors of less than three or even not
significantly in rec10-109 (Table S1; DeVeaux and Smith, 1994;
Davis et al., 2008; Estreicher et al., 2012). Although these differ-
entials were initially described as ‘‘region specific,’’ our data
suggest that they are ‘‘site specific,’’ because no large region
of the mutant genomes retains all hot spots present in wild-
type (lab website). We note, however, that DSB hot spots are
nearly eliminated in these mutants on chromosome 3, on which
the largest reductions in recombination are observed (Table S1;
DeVeaux and Smith, 1994; Ellermeier and Smith, 2005; Davis
et al., 2008; Estreicher et al., 2012).
We infer that the residual recombination in these four mutants
reflects mostly non-hot spot DSBs with some contribution from
residual DSBs at hot spots (see below). The rec10-109 missense
mutant protein may have diminished ability to bind Rec25,
Rec27, or Mug20 (or their complex) but retained the ability to
activate Rec12 for DSB formation in DSB-cold regions; this
hypothesized feature would account for the rec10-109 pheno-
type being similar to that of rec25D, rec27D, and mug20D
Rare DSB Hot Spots Partially Independent of Cohesins
and LinE Proteins
In null mutants lacking any one of these proteins other than
Rec10, we discovered that DSBs still occur at some hot spots
(roughly 10% of the total), although the frequency of DSBs at
these sites is reduced (Figure 3; Ellermeier and Smith, 2005;
Davis et al., 2008). rec10D lacks essentially all DSBs (Figure 3).
Therefore, Rec10 can activate Rec12 at these few hot spots
without the other proteins. What distinguishes these hot spots
fromthe majority remainsunknown, but it may reflect the meiotic
transcription pattern: many of the residual hot spots are next to
genes with large meiosis-specific 50UTRs (unpublished data).
Alternatively, Rec10 may bind these sites in a manner that allows
Relation to Higher-Order Chromatin Structure and
Meiotic Recombination in Other Species
Our data contrast sharply with related observations
S. cerevisiae, the only other species in which meiotic DSBs
no other proteins have been shown to define DSB hot spots
genome-wide with the high enrichment shown by Rec25,
Rec27, and Mug20. Other S. cerevisiae proteins, notably in-
cluding Rec8, preferentially bind to DSB-cold regions, although
the degree of anticorrelation of Rec8 binding and DSBs is
much less (R2= 0.068 [Glynn et al., 2004], 0.036 [Panizza
et al., 2011], or 0.14 [Pan et al., 2011]) than the degree of corre-
lation of Rec27 binding and DSBs (R2= 0.59; Figure 4). The
Protein Determinants of Meiotic DSB Hot Spots
Molecular Cell 49, 983–996, March 7, 2013 ª2013 Elsevier Inc. 993
binding of other S. cerevisiae axial element proteins also weakly
anticorrelates with DSBs: for Red1 and Hop1, R2= 0.068 and
0.04, respectively (our analysis of the protein data of Panizza
et al.  and the DSB data of Bu ¨hler et al. ). Thus,
although the binding of Rec27 (positively) accounts for about
60% of the DSB distribution, the binding of S. cerevisiae Rec8,
Red1, and Hop1 (negatively) accounts for only about 10% of
the DSB distribution.
In S. cerevisiae the anticorrelation of DSBs and binding of
Scc1, the mitotic paralog of Rec8, and Red1 led Blat et al.
(2002) to propose that DSBs form in chromatin loops but are re-
paired when that site is on the axis, because plant and animal
DSB-repair protein Rad51 foci are on the axis. Panizza et al.
(2011) and Miyoshi et al. (2012) observed that Spo11 (Rec12)
partner proteins bind to the axis and interpreted their data in
the same framework: DSB sites in the loops are brought to
the axis before DSB formation and subsequent repair. Our
data provide a different paradigm: the DSB-activating LinE
(axial) proteins Rec25, Rec27, and Mug20 bind directly at or
near the sites where DSBs are later made, perhaps by
activating Rec12 bound nearby. That meiotic chromosome
dynamics are different in these two yeasts is also illustrated
by themicroscopiclines (axial
S. cerevisiae but dozens of dots of Rec8 in S. pombe (e.g.,
Ding et al., 2006; Davis et al., 2008). Thus, the mechanism by
which Rec8, for example, promotes DSB formation apparently
differs in the two yeasts.
Role of Hot Spot-Binding Proteins in Crossover Control
Crossover formation is carefully controlled, presumably to
ensure proper homolog segregation at the first meiotic division
(Figure 1), and numerous aspects of crossover control have
been described (Phadnis et al., 2011). In S. pombe crossovers
are much more evenly distributed across the genome than are
DSBs, a feature called crossover invariance (Figure 7; Hyppa
and Smith, 2010). At hot spots DSBs are repaired pre-
dominantly with the sister chromatid, which cannot yield a
crossover, whereas in cold regions DSBs appear to be re-
paired primarily or exclusively with the homolog and yield
a crossover in about 80% of repair events (Cromie et al.,
2005; Hyppa and Smith, 2010). The mechanism of this partner
choice for DSB repair presumably reflects some feature of the
chromosome before DSB formation, because otherwise we
suppose that, once formed, a DSB at one site is like a DSB
at any other site.
Partner choice may reflect the presence of Rec25, Rec27, and
Mug20 almost exclusively at DSB hot spots (Figure 3A), effec-
tively establishing domains of differential repair, because no
other chromatin-associated pre-DSB proteins are known to
distinguish the majority of hot spots from non-hot spots. The
following observations support this hypothesis. In rec8D
mutants, residual DSB frequencies are proportional to the
enrichment of bound Rec27 (Figure 5), suggesting that these
DSBs are Rec27-dependent, but residual recombination is not
Rec25 dependent (rec25D and rec27D single and double
mutants are indistinguishable, suggesting that Rec25 and
Rec27 act together [Davis et al., 2008]). Thus, the Rec27-depen-
dentDSBsinrec8Dmutants apparentlydonotgiveriseto cross-
overs, perhaps because they are repaired by interaction with the
sister chromatid (Hyppa and Smith, 2010). Dmc1 strand
exchange protein is not required for DSB repair at strong hot
spots and plays a larger role in recombination in DSB-cold
regions than at DSB hot spots (Hyppa and Smith, 2010).
Rec25, Rec27, and Mug20 may therefore prevent Dmc1 from
acting at hot spots. Rad51, a paralog of Dmc1, acts both in
DSB-cold regions and at hot spots (Hyppa and Smith, 2010)
and, in this view, is immune to inhibition by Rec25, Rec27, and
Mug20. Perhaps Dmc1 has an intrinsic preference for repair
with the homolog when it can act. Regardless of these consider-
ations, the requirement for Rec25, Rec27, and Mug20 for DSB
formation at hot spots and for most crossovers implicates these
proteins in crossover control by determining both the spatial
position and the break frequency of DSB hot spots and perhaps
their mode of repair as well.
S. pombe Strains and Culture Conditions
S. pombe strains, genotypes, and sources of alleles are listed in Table S3.
Diploid pat1-114 strains were thermally induced for meiosis and analyzed
for DNA content by flow cytometry as described by Cervantes et al. (2000).
Meiotic crosses were conducted and analyzed as described by Young et al.
HotspotHotspot DSB cold regionDSB cold region
DSB frequencyDSB frequency
Crossover invariance (0.16 cM/kb)Crossover invariance (0.16 cM/kb)
Mostly no genetic
despite DSB hotspots
Figure 7. Proposal for Crossover Control by Rec25-Rec27-Mug20
Rec10 (blue balls) binds across chromosomes, enriched at DSB hot spots.
Rec25-Rec27-Mug20 complex (red oval) binds hot spots and activates Rec12
to make high-frequency DSBs and biases DSB repair toward the sister, giving
a low crossover:DSB ratio. In DSB-cold regions repair is biased toward the
homolog, giving a high crossover:DSB ratio. The result is a nearly uniform
distribution of crossovers across the genome (crossover invariance; Hyppa
and Smith, 2010).
Protein Determinants of Meiotic DSB Hot Spots
994 Molecular Cell 49, 983–996, March 7, 2013 ª2013 Elsevier Inc.
Diploid pat1-114 cells induced for meiosis were examined for a protein fused
at its C terminus to GFP as described by Davis et al. (2008). Cells were fixed
induction of meiosis. Signals were similar to those in unfixed cells, except for
cells. Details of these methods and those for nuclear spreads are in Supple-
mental Experimental Procedures.
Genome-wide DSB Frequency and Protein Localization
DSB frequencies across the genome were determined by hybridization of
DNA PCR-amplified from Rec12-DNA covalent linkages in diploid pat1-114
rad50Scells 5hrafter beinginducedfor meiosis(Cromie etal.,2007).Proteins,
as GFP or FLAG fusions, were assayed across the genome by crosslinking
proteins and DNA with formaldehyde 3.5 hr after meiotic induction of pat1-
114 cells. Further details are in the Supplemental Experimental Procedures.
Tiling data in this study have been deposited in the NCBI Gene Expression
Omnibus under accession number GSE43122 and are available in graphic
Supplemental Information includes nine figures, three tables, Supplemental
Calculation, and Supplemental Experimental Procedures and can be found
with this article online at http://dx.doi.org/10.1016/j.molcel.2013.01.008.
We are especially grateful to Anna Estreicher and Josef Loidl for strains with
mug20 mutations and unpublished information. We thank Emily Higuchi and
Nishka Mittal for data in Table S1; Randy Hyppa and Naina Phadnis for strains;
Luisa Bustamante-Jaramillo, Daniel Go ´mez-Sa ´nchez, Jeff Delrow, and Gareth
Cromie for assistance; Monica Colaia ´covo for fruitful discussions; and Sue
Amundsen, Michael Lichten, Naina Phadnis, Walt Steiner, and Sarah Zanders
for helpful comments on the manuscript. This work was supported by NIH
grants GM031693 and GM032194 (to G.R.S.) and grant FEDER-BFU2010-
14954 from the Spanish Ministry of Science and Innovation (to C.M.-C.).
S.G.-V. was supported by the JAE-Tech CSIC program. The Instituto de
Biologı ´a Funcional y Geno ´mica acknowledges support from the Ramo ´n
Received: August 21, 2012
Revised: November 15, 2012
Accepted: January 3, 2013
Published: February 7, 2013
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