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TRENDS in Genetics Vol.19 No.9 September 2003
Distribution of meiotic recombination
sites
Bernard de Massy
Institut de Génétique Humaine, 141 rue de la Cardonille, 34396 Montpellier cedex 5, France
Meiotic recombination generates gene conversion and
crossover events that are distributed heterogeneously
in the genome. Studies in yeast show that initiation of
recombination, which occurs by the formation of DNA
double-strand breaks, determines the distribution of
gene conversion and crossover events that take place in
nearby intervals. Recent data in humans and mice also
indicate the presence of highly localized initiation sites
that promote crossovers clustered around the region of
initiation and seem to share common features with
sites in yeast. On a larger scale, chromosomal domains
with various recombination rates have been identified
from yeast to mammals. This indicates a higher level of
regulation of recombination in the genome with potential consequences on genome structure.
During meiosis, recombination between homologous chromosomes generates gene conversion (nonreciprocal events)
and crossing over (reciprocal events). Analyses in many
organisms show that meiotic recombination events are not
distributed randomly in the genome. Given that homologous recombination can, in principle, spread over large
distances, the distribution of events depends on where
recombination is initiated as well as how far it extends.
The combination of genetic and molecular analysis in
Saccharomyces cerevisiae and, more recently, in mammals
shows that recombination events (gene conversion and
crossover) cluster tightly in the vicinity of initiation
sites. Therefore, the distribution of exchanges essentially
reflects the distribution of initiation sites. Here, I review
the properties of these sites and associated events in the
yeasts S. cerevisiae, and Schizosaccharomyces pombe,
mice and humans.
The current model for meiotic recombination
The current model of meiotic recombination combines
the double-strand-break repair (DSBR) model [1] and
the synthesis-dependent strand-annealing (SDSA) model
(Box 1) (reviewed in [2]). The combination of these pathways has been proposed to explain extra-chromosomal
recombination in mammalian cells [3] and human
minisatellite rearrangements [4]. Molecular analysis in
S. cerevisiae provides direct support for this model with
respect to meiotic recombination [5,6].
Several specific features of this model are particularly
relevant for the comparative analysis of events in yeast
and mammals presented below. First, the chromatid that
Corresponding author: Bernard de Massy (bdemassy@igh.cnrs.fr).
initiates and, therefore, where the DSB occurs, is the
recipient of genetic information. This leads to conversion
on the initiating chromatid. Conversion occurs by mismatch repair, and not by double-strand gap as originally
proposed. Second, the strand exchange is initiated and
extended from the 30 ends, thus, heteroduplex forms
initially in regions where degradation of 50 ends occurs
but can extend further by either strand displacement or
branch migration of Holliday junctions. Repair of heteroduplex produces either gene conversion or restoration
depending on the choice of the corrected strand. If Holliday
junctions are resolved within the interval defined by the
gene-conversion tract, the sites of crossing over map at
the boarders of these tracts. However, branch migration
of the Holliday junctions can lead to crossover away from
this region.
Local distribution of recombination in yeasts
DSBs, which are detected by molecular analysis, are
generated by Spo11 in conjunction with several other
proteins at many sites in the genome (reviewed in [7]).
Four levels of constraint have been observed with respect
to the distribution of DSBs in yeast: (1) at the nucleotide
level, DSBs are not distributed randomly, but no clear
sequence motif has been identified; (2) DSBs are commonly
located in accessible regions of the chromatin; (3) the
formation of DSBs appears to depend on chromosome
organization and to occur preferentially in chromatin loops
away from DNA-axis associations that are mediated by
cohesins and Red1 [8,9]; and (4) DSBs are clustered in
chromosome domains and are reduced in telomeric regions
and the vicinity of centromeres [10,11].
Local distribution of initiation sites
DSBs occur in highly localized regions and spread over
70–250 bp (reviewed in [7]). DNA sequence analysis reveals
no unique conserved consensus sequences, although a
degenerate 50-bp motif partly correlates with DSB sites.
However, one common feature is that DSBs are located in
accessible regions of the chromatin next to either promoters or binding sites for transcription factors (reviewed
in [12– 14]). Based on two studies, DSB activity does not
correlate with local transcriptional activity, but depends
on transcription-factor binding (HIS4 in S. cerevisiae and
ade6-M26 in S. pombe). The constraint imposed by chromatin is also revealed by the activity of a Gal4–Spo11 fusion
protein, which induces high DSB levels in a region that
contains a Gal4-binding site and normally a low DSB
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515
TRENDS in Genetics Vol.19 No.9 September 2003
Box 1. Double-strand-break repair (DSBR) and synthesis-dependent strand-annealing (SDSA) models of meiotic
recombination
Two interacting duplexes from homologous chromosomes are shown
as black and red lines in Fig. I . Sister chromatids present at this stage
are not represented. Initiation occurs by double-strand break (DSB)
formation on the red chromatid. DSB ends are then degraded (Fig. Ia) to
generate long, 300 single-strand tails of several hundred base pairs. This
generates a gradient of single-strandedness in a population of cells,
with a higher level proximal to the break [77]. One single-strand invades
the homologous duplex (Fig. Ib) to form a heteroduplex and a D loop
that is extended by DNA synthesis. This has two possible outcomes.
In the DSB repair (DSBR) pathway, the D loop captures the second
30 end (Fig. Ic). DNA synthesis and ligation generate a double Holliday
junction [78 –80] with heteroduplex DNA flanking the DSB site [81]. This
recombination intermediate is resolved by the cleavage and ligation of
strands of same polarities at identical positions, which generates
crossover products. This pathway might also generate noncrossovers
(not shown). Mismatch repair of heteroduplexes can lead to either gene
conversion or restoration depending on the choice of the corrected
strand. Here, repair towards gene conversion is shown on both sides of
the initiation point. This is the major direction of repair for markers
located near the DSB site, probably because of the preferential removal
of strands with an available 30 end (i.e. strands from the initiating
duplex). Repair of heteroduplex DNA away from the DSB site might take
place on the opposite strand and lead to restoration.
In the synthesis-dependent strand-annealing (SDSA) pathway, the
D loop is disassembled by displacement of the newly synthesized
strand, which anneals with the other DSB end (Fig. Id). Repair of the
break is completed by DNA synthesis and ligation. Mismatch repair of
heteroduplex DNA generates gene conversion without crossover.
In both pathways, the process of mismatch repair is presented
arbitrarily at the last step. This is probably an oversimplified view
because mismatch repair itself might involve several pathways and act
at more than one step during the process [80]. Various alternatives
could be considered: for example, early repair of heteroduplex DNA at
the stage of D-loop formation could lead to conversion events on both
sides of the initiation event through the SDSA pathway. Little is known
about how and when Holliday junctions are processed, and when
mismatch repair takes place, although some data indicate that these
two events might be coordinated [82]. In the case shown, Holliday
junctions do not migrate from the region of initial heteroduplex DNA,
therefore crossover molecules have exchange points at the ends
of conversions tracts (shown as flags). More distal crossover points
might form if one or both Holliday junctions migrated away from the
initiation site.
Although most data that support this model result from studies
in yeast, the conservation of the major proteins involved indicates
that the main lines should be conserved in yeast and mammals [83].
DSB formation
3′
5′
3′
5′
(a)
5′ to 3′ resection
Heteroduplex
Strand-invasion, D-loop
formation, DNA synthesis
(dotted line)
(b)
DSBR
SDSA
Strand-displacement
Second end capture,
synthesis, ligation
(c)
(d)
Strand annealing,
synthesis, ligation
Holliday junction
resolution
Mismatch repair
Mismatch repair
Crossover
point
Crossover point
Gene conversion
tract
Gene conversion
with crossover
Gene conversion
tract
Gene conversion
without crossover
TRENDS in Genetics
Fig. I. Double-strand break repair (DSBR) and synthesis-dependent strand-annealing (SDSA) pathways. Reproduced with permission from [5].
activity [15]. However, some DSB sites do not seem to be
associated with the binding of transcription factors,
indicating that there might be alternative determinants
for accessibility to the recombination machinery. It has
been proposed that a chromatin-modification code contributes to the definition of DSB sites [13].
http://tigs.trends.com
Distribution of gene-conversion events
The presence of highly localized initiation sites that are
separated by regions that contain few detectable initiation
events allows the measurement of how recombination
extends from the initiation site. At several loci, genetic
analyses show that gene conversion frequencies decrease
516
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TRENDS in Genetics Vol.19 No.9 September 2003
Table 1. Analysis of gene-conversion gradients in yeasts and of a crossover gradient in mice
Schizosaccharomyces
pombe
Saccharomyces cerevisiac c
Highest markera
Lowest marker
Distanceb
P
Linear correlation within
99% confidence (P , 0.01)
Mean tract length (bp)
95% confidence interval
Mus musculus
ARG4 (1)
ARG4 (2)
HIS2
HIS4 (1)
HIS4 (2)
ade6
ura4-aim
Psmb9
1
0.02
1383
0.9975
Yes
1
0.12
2100
0.999
Yes
1
0.33
879
0.9988
Yes
1
0.44
2270
0.9997
No
1
0.19
1592
0.999
No
1
0.05
2357
0.9987
Yes
1
0.33
601
0.9987
Yes
1
0.03
790
0.9958
Yes
794
667 –981
2078
1420–3860
1679
1173– 2949
6562
Not determined
1963
Not determined
1591
1421 –3862
1484
946–3442
480
315 –1018
a
At each locus, the frequency of events for the highest frequency marker is normalized to 1.
Distance in base pairs between the highest and lowest frequency markers. The frequency of events as a function of distance follows an exponential law ( p value). References:
ARG4 (1), [60]; ARG4 (2), [18]; HIS2, [61]; HIS4 (1), [62]; HIS4 (2), M.F.F. Abdhulla and R.H. Borts, pers. commun.; ade6, [19]; ura4-aim, J. Kohli, pers. comm.; Psmb9, [35].
c
(1) and (2) refer to two independent experiments.
b
http://tigs.trends.com
co-conversion with a proximal promoter marker indicates
the presence of distal initiation events [17].
In agreement with the DSBR model and the processivity of heteroduplex repair, analysis at ARG4 and ade6
also show that most conversion tracts are continuous
[18,19]. However, discontinuous conversion tracts are
observed in other fungi, such as Neurospera crassa
[20,21] and Ascobolus immersus [22], which indicates
either alternative intermediates or alternative processing
of intermediates by the mismatch-repair machinery in
these species.
Distribution of crossing over
The model of DSBR allows alternatives to how and where
Holliday junctions could be resolved, and mapping geneconversion events does not directly address this issue.
There are at least two possibilities in terms of location:
either Holliday junctions are resolved in the area covered
by the conversion tracts or they migrate further away in
flanking regions. It should be emphasized that the location
of crossovers might be distinct from the actual point of
Holliday-junction resolution. Two studies performed in
S. cerevisiae, provide more information about this issue.
Symington et al. [23,24], used polymorphisms to select and
map crossovers in a 22 kb interval and two regions that
contained high densities of crossover were identified.
(a)
(b)
1.0
0.0015
0.8
Frequency
Relative frequency
on both sides of the initiation site. The molecular mechanism responsible for this decrease, referred to as a
gradient of gene conversion, is debated. It could involve a
regulation of the directionality of mismatch repair, which
influences the relative frequencies of gene conversion and
restoration as a function of the distance from initiation, or
it might involve a regulation of heteroduplex length, or
both (Box 1). Data from several loci are reported in Table 1.
At each locus gene conversion frequencies of several
markers were measured. In each case, meiotic DSBs
were detected and the marker located closest to the DSB
site was found to have the highest frequency of gene
conversion. The relative frequencies of only the highest
(normalized to 1) and lowest markers are indicated in
the table.
When all markers tested are taken into account, the log
of the frequencies of gene conversion follows a linear
relationship as a function of distance with a 99% confidence interval in all cases tested except the HIS4 locus.
This exponential relationship is best explained by assuming that repair events that lead to gene conversion travel
from the initiation point with a fixed probability (1 2 p) of
stopping at a given position (n). The probability that a gene
conversion tract extends over a length L to position n from
initiation is: PðL ¼ nÞ ¼ pn ð1 2 pÞ ¼ (probability of reaching position n) £ (probability of stopping at position n).
Assuming independent processing of heteroduplexes on
either side of the DSB, as proposed in the DSBR model,
the length distribution of conversion tract is: PðCo ¼ n ¼
ðn þ 1Þpn ð1 2 pÞ2 ; with a the mean of (1 þ p)/(1 2 p) [16].
Examples of the distributions of gene-conversion frequencies and gene-conversion-tract lengths at the ARG4
locus are shown in Fig. 1. Applying this to other loci results
in a fairly homogeneous range of gene-conversion-tract
lengths in S. cerevisiae (except at the HIS4 locus) and
S. pombe. The means range from 800– 2000 bp at most loci.
At the HIS4 locus, the distribution of gene conversion is
not well fitted by an exponential law and the significance
of the predicted mean is therefore questionable. This
might reflect either some specific property of recombination in this region or that some of the events detected are
not initiated from DSBs located in the HIS4 promoter
region. The observation that a high proportion (, 50%)
of conversion events at a distal marker do not involve
0.6
0.4
0.2
0
0
500 10001500200025003000
Distance (bp)
0.0010
0.0005
0
0
500 1000 1500 2000 2500 3000
Distance (bp)
TRENDS in Genetics
Fig. 1. (a) Distribution of gene conversion frequencies at the ARG4 locus. Values
are normalized to 1 for the marker with the highest frequency set at position 0.
Experimental values for eight markers are shown in pink. The best fit of the linear
correlation between the log of frequencies and distance is shown in blue
( p ¼ 0.99748). PðL $ nÞ ¼ p n : (b) Distribution of the gene-conversion tract length
assuming a bidirectional processing on either sides of the initiation point and an
exponential law of p ¼ 0.99748. PðCo ¼ nÞ-ðn þ 1Þp n ð1 2 pÞ2 : The mean of this
distribution is 794 bp.
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517
TRENDS in Genetics Vol.19 No.9 September 2003
DSBs were analyzed. Subsequent DSB analysis in this
region showed that the two regions with high crossover
density also have high levels of DSBs [25]. From the
sizes of the interval tested, it is likely that most crossovers occur within , 2 – 3 kb of their initiation site. Most
gene-conversion tracts detected are ,2 kb long and are
continuous. A minority (8%) of long conversion events
(4.5–12.0 kb) were also identified. Using a similar approach,
Borts et al. mapped gene conversion and crossing-over
association. As above, crossover mapped to the boarders
of conversion tracts in most cases, but positions of the
DSBs were not determined [26,27]. These data are consistent with a model in which the Holliday junctions do
not migrate from the region of initial asymmetric heteroduplex (Box 1).
By contrast, a recent comparison of DSB and crossover
density in large intervals in the S. pombe genome led
Young et al. [28] to propose that Holliday junctions migrate
and are resolved at long distances from their site of
initiation in this organism. However, in a study of gene
conversion and crossover association at the ade6 locus,
crossover separated from selected conversion events were
detected in one interval and found to represent only a
minority of events [19]. Further analyses are therefore
needed to determine the proportion of events that follow
this pathway in S. pombe [29].
Local distribution of recombination events in mammals
Local distribution of sites
Several recombination hot spots have been defined in
humans and mice. These are based on the higher density of
crossing over (relative to either adjacent regions or the
genome average) identified from pedigree analysis in
humans and screening recombinant in-crosses between
laboratory mouse strains. More recent studies have also
identified meiotic recombination hot spots and highresolution mapping of recombination events through
direct molecular analysis in humans and mice. The level
of precision, therefore, differs according to the methods
used. Table 2 summarizes those that are precisely located
on the physical map. Large differences are observed
between the activity of these hot spots, and their definition
is obviously relative. For example, recombination at Dna1
is three-fold lower than the genome average but about
seven-fold higher than surrounding regions.
In terms of location, of the 17 hot spots listed, nine are
intergenic (50 and 30 of genes), three are within introns, and
five are mapped at insufficient resolution to distinguish
intronic and exonic locations. Although analysis of DNA
sequences in these regions has identified no obvious
motifs, several properties have been noted [30,31]. Even
though not confined to transcription-promoter regions,
determinants for hot-spot localization in mammals might
Table 2. Hot spots in humans and mice
Hot spota
Frequency
(cM)
Interval
(Kb)
Fold above
genome
average
Location
Methods
Human
MS32
0.04
1.5
30
Intergenic, next to minisatellite
CEB1
0.16
2
$ 70
Intergenic, next to minisatellite
b-Globin
0.9
11
90
Intergenic, 50 of b globin
PGM1
CMT1a
1.7
0.0015
58
1.4
30
1.2
Intragenic, intron or exon
Intergenic
DNA1
0.0005
1.9
0.3
Intergenic, 50 of DNA
DNA2
0.0037
1.3
3
Intergenic, 30 of DNA, next Alu
DNA3
0.13
1.2
120
Intergenic, 30 of DNA, next Alu
DMB1
0.0031
1.8
2
Intragenic, intron or exon
DMB2
0.028
1.2
30
Intergenic, 30 of DMB
TAP2
0.0058
1
10
Intragenic, intron
PAR1
0.3
3.9
90
Intragenic, intron or exon
Pedigree analysis and direct
molecular detection
Pedigree analysis and direct
molecular detection
Pedigree analysis and single
sperm analysis
Pedigree analysis
Pedigree analysis and direct
molecular detection
Pedigree analysis and direct
molecular detection
Pedigree analysis and direct
molecular detection
Pedigree analysis and direct
molecular detection
Pedigree analysis and direct
molecular detection
Pedigree analysis and direct
molecular detection
Pedigree analysis and direct
molecular detection
Pedigree analysis and direct
molecular detection
Mouse
Ea
Eb
Psmb9
0.9
0.1
1
0.4
4
1.8
3750
40
930
Intragenic, intron
Intragenic, intron
Intergenic, 30 of Psmb9
Gc7
Pb
Unknown
1.5
2.4
5.3
Unknown
470
Intragenic, intron or exon
Intragenic, intron or exon
a
Genetic crosses
Genetic crosses
Genetic crosses and direct
molecular detection
Genetic crosses
Genetic crosses
Locus Link databaseb
HBB; locus ID, 64162
PGM1; locus ID, 5236
CMT1A; locus ID, 1248
HLA-DOA; locus ID, 3111
HLA-DOA; locus ID, 3111
HLA-DOA; locus ID, 3111
HLA-DMB; locus ID, 3109
HLA-DMB; locus ID, 3109
Tap2; locus ID, 6891
Shox; locus ID, 6473
H2-Ea; locus ID, 14968
H2-Eb1; locus ID, 14969
Psmb9; locus ID, 16912
D17H6S56E-3; locus ID, 27762
H2-Pb; locus ID, 15004
For each hot spot, the frequency of crossing-over is given in cM in the interval that defines the hot spot. The increase relative to the genome average is calculated taking a
value of 0.89 cM Mb21 for human [63] and 0.6 cM Mb21 for mouse [64]. These are average values for male and female recombination rates. The Cmt1a hot spot is a preferred
site for unequal exchanges. The methods used to define the locations and frequencies of the hot spots are indicated. References: MS32, [32]; CEB1, [4]; b-globin, [65– 67];
Pgm1, [68]; Cmt1a, [69]; Dna1, Dna2, Dna3, Dmb1 and Dmb2, [33]; Tap2, [30]; Par1, [70]; Ea, [71]; Eb, [72,73]; Psmb9, [35,74,75]; Gc7, [76]; Pb [31].
b
http://www.ncbi.nlm.nih.gov/LocusLink
http://tigs.trends.com
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TRENDS in Genetics Vol.19 No.9 September 2003
share common features with those described in yeast, such
as chromatin accessibility. To what extent gene-regulatory
elements and transcription factors play a role in this
localization is currently unknown.
Local distribution of events
Direct molecular detection of recombination events was
developed by A. Jeffreys group to study minisatellite
rearrangements that take place at a high rate during
meiosis. This strategy allows the selective amplification of
recombinant molecules by PCR. In minisatellite regions,
the analysis of events either with or without the exchange
of flanking markers indicated the existence of two pathways, the DSBR and SDSA pathways described in Box 1.
In addition, extensive polymorphisms have allowed the
mapping of minisatellite rearrangements that show the
presence of gene conversion and crossover gradients [4,32].
The same approach has been used to study some crossover
hot spots that are not associated with minisatellites in
humans and mice (Table 2). Although the activities of
these hot spots differ by up to several-hundred-fold, they
share common features, such as clustering of crossovers in
a small interval (1 –3 kb) and nonrandom distribution with
a central region of highest density. In addition, all recombinant molecules detected are simple exchanges with no
mosaicism.
The distribution of crossovers at the mouse Psmb9 hot
spot is presented in Fig. 2. The properties of Psmb9 are
compatible with the presence of an initiation site that
induces recombination events that are repaired according
to the DSBR model, and where crossover points correspond
to the borders of gene-conversion tracts (Box 1). The
distribution of crossovers fits an exponential law on either
side of the central region, corresponding to a mean value
for tract sizes of 480 bp, which is slightly shorter than that
deduced from gene-conversion events in yeast (Table 1).
This observation of highly localized crossovers fits with
the analysis of linkage disequilibrium near hot spots in
humans. In the regions between hot spots [e.g. the 100 kb
interval between Dmb and Tap2 (Fig. 3a)], there is a strong
linkage, which indicates that few crossovers extend outside the hot-spot regions [33]. Two additional pieces of
information strongly support these hot spots as sites of
initiation. First, one prediction of the DSBR model is that
when only one homologous chromosome initiates, of the
two types of crossover molecules that can be recovered,
the exchange point of one will always be on one side of
the initiation site, and the other on the opposite side
(Box 1). In some heterozygous situations, such a bias has
been observed at the human Dna2 hot spot [34] and at the
mouse Psmb9 hot spot (F. Baudat and B. de Massy, pers.
commun.). Second, the presence of an initiation site
predicts a high frequency of gene-conversion events that
are not associated with a crossover. Recombinant molecules compatible with such events occur at the Psmb9
hot spot for a marker located in the central region. The
sizes of the gene-conversion events (without crossover
in the interval tested) recovered in this analysis were
short (, 500 bp), which is compatible with the analysis of
crossover distribution discussed above [35].
http://tigs.trends.com
Crossover frequency (cM Mb–1)
518
1500
210 bp
1000
500
100
T/C T/C
TaqI
T/C T/A
BsrFI StyI
(A)8/9
SphI
Eco57I
TRENDS in Genetics
Fig. 2. Distribution of crossovers at the Psmb9 hot spot in mice. Recombinant
molecules were selected by allele-specific PCR using primers 3 kb apart. All events
detected map within the TaqI –Eco57I interval (1.8 Kb) in which the average polymorphism is 0.65%. The crossover frequency relative to distance (cM Mb21) is
given for the intervals analysed, based on the analysis of 69 independent events
(38 and 31 of each type of exchange). The interval with the highest crossover density is defined by the 210-bp BsrFI– StyI region and corresponds to the center of
the initiation site. The T ! C, T ! A and (A)8/9 polymorphisms are indicated. Data
reproduced with permission from [35].
Global distribution of recombination in yeasts
The extensive molecular analyses in yeasts reveal that
DSB sites cluster into chromosomal domains. For instance,
on S. cervisiae chromosome III (300 kb long), two separate
domains (,50 kb each) with high DSB levels can be
identified [10,11]. The influence of domains is well illustrated by the correlation between the level of DSB of an
inserted sequence and the global DSB level of the domain
where it is inserted [36]. Components that define these
domains are unknown. In addition, centromeric regions
and telomeres (10 – 20 kb proximal) are repressed for DSB
formation.
Global distribution of recombination in mammals
High-resolution analysis of a 200 KB interval in the major
histocompatibility complex (MHC) class II region reveals
six hot spots (Table 2), the activities of which varies from
0.3 to 110 cM Mb21. These hot spots cluster in three
regions and are separated by 50 – 100 kb (Fig. 3a). In these
intervals, the values of recombination frequencies are a
rough estimate based on linkage disequilibrium (LD)
analysis. The crossovers in the hot-spot regions, which
cover , 10% of the whole interval, account for . 95% of
crossover events. This analysis illustrates clearly the high
discontinuity of distribution of recombination rates and a
surprisingly high level of variation in recombination rates
between hot spots (up to 400 fold).
At a lower resolution, crossover distribution in the
whole MHC (3.5 Mb) was analyzed using pedigree and
single-sperm analysis [37]. Markers 30 –200 kb apart
define 30 intervals in which crossover density varies
from 0.15 to 2.50 cM Mb21 with an average of
0.49 cM Mb21. This is slightly lower than the male average
(0.85 cM Mb21 from [38]) (Fig. 3b). Six regions are ‘hotter’
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TRENDS in Genetics Vol.19 No.9 September 2003
(a)
1000
cM Mb–1
100
10
1
3 mcM
0.1
3 mcM
4 mcM
?
0.01
(b)
3.0
Frequency (cM Mb–1)
0
2.5
50
100
kb
150
TAP2
PSMB9
TAP1
PSMB8
DMA
DMB
DNA
RING3
0
200
centromeric and telomeric regions are, on average, systematically cold and hot, respectively. However, there are large
sex differences in these regions, with, in general, a higher
frequency of recombination near centromeres in females
and a higher frequency of recombination near telomeres in
males. Although the average recombination rate in females
is 1.65 times higher than in males, some regions in chromosome arms can be hotter in males than females. In fact,
the correlation (R value) between male and female rates
is 0.57, which indicates that a large amount of the control
of the distribution of recombination along chromosomes
is regulated by factors other than the primary DNA
sequence [38]. These chromosome-wide variations are also
described at the cytological level in humans and mice,
through the analysis of chiasmata and late recombination
nodules [42,43].
2.0
1.5
1.0
0.5
0
0
500
1000
1500
2000
2500
3000
3500
Distance (Kb)
Class II
(c)
Chromosome 6
3
Frequency (cM Mb–1)
519
2
1
0
c
MHC
0
50
100
Mb
150
200
TRENDS in Genetics
Fig. 3. Recombination hot spots on human chromosome 6. (a) High-resolution
mapping of crossover within 200 kb in the major histocompatibility complex
(MHC) class II region. Recombination frequencies were measured by direct
molecular detection in the six intervals showing peaks of recombination rates
(Dna1, Dna2 and Dna3 near the DNA gene, Dmb1 and Dmb2 near the DMB gene
and Tap2). These values are from male meiosis. Between these sites, recombination rates were approximated from linkage disequilibrium studies. (b) Mapping
crossovers in the MHC region by sperm typing. Each point represents the centre of
the intervals (average of 12 individuals). The position of the 200 kb interval of the
class II genes in (a) is shown as a solid line, the remainder of this region is shown
as a dotted line. (c) Mapping crossovers on human chromosome 6 (male and
female). Data are plotted with 3 Mb windows, moved by 1 Mb steps. The positions
of the MHC and the centromere (c) are shown. Data reproduced with permission
from: (a), [33]; (b), [37]; and (c), [38].
than average, one of which includes the strongest hot-spot
defined at high resolution (Dna3). At still lower resolution,
the human and mouse genetic maps reveal Mb fluctuations in recombination rates [38 – 41]. These variations
are shown for human chromosome 6, which contains the
MHC region (Fig. 3c). On this chromosome, recombination
rates vary from 0.1 to 3.0 cM Mb21 with an average of
1 cM Mb21, which is close to the genome average in this
study (1.13 cM Mb21). It is likely that these variations
are caused by a combination of variations in density and
levels of activity of initiation sites in these intervals. The
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GC content and the distribution of recombination
The analysis of DSB distribution in S. cerevisiae shows a
correlation between DSB activity and GC content: domains
with high levels of DSB correspond to GC-rich domains
[10,11]. Furthermore, DSB activities of a DNA fragment
inserted at various locations on yeast chromosome III
correlate with local GC content [44]. In humans, two LD
studies indicate a correlation between high GC content
and elevated recombination rates [45,46]. However, estimation of recombination rates based on LD analysis is
subject to several caveats and should be taken with caution
[47]. Nevertheless, the correlation between recombination
density and GC is observed from the complete human
genetic map [48,49], and has also been deduced from
immunolocalization of recombination nodules [50]. GC-rich
regions are organized into chromosome domains, also
identified as isochores [51], which correspond to gene-rich
R bands. Many properties distinguish GC-rich from GC-poor
domains, such as replication timing and the frequency of
repeated sequences [52,53]. It is important to note that
although the correlation is significant, the variation in
GC content accounts for only a small part of the variation
in recombination rates (17%, R2 ¼ 0.17) [38]. The detailed
analysis of recombination in the human MHC show no
correlation between recombination rate and GC content:
the sharp increase in the proportion of GC residues in class
II compared to class III genes does not cause a difference in
recombination rates on either side of this transition
(0.66 cM Mb21 in the class II region, intervals Dpb1 to
Notch4, and 0.46 cM Mb21 in the class III region, intervals
Notch4 to MicB) [37]. Furthermore, in the MHC class II
region, of the three crossover clusters that have been
mapped at high resolution (Dna3, Dmb2 and Tap2), only
one (Tap2) corresponds to a region with a higher than
average GC content. In this region, three GC peaks are
located in cold intervals for recombination (B. de Massy
and L. Duret, unpublished). Given that these hot spots are
in regions with relatively low GC content, one can conclude
that a high level of recombination activity does not require
a GC-rich domain or a high local GC content. The correlation observed at high resolution in yeast is, therefore, not
universal.
Whatever the correlation observed, it is important to
determine whether it is either a cause or a consequence
520
Review
TRENDS in Genetics Vol.19 No.9 September 2003
(or both) of the recombination activity. Several studies
address this issue, taking into account several parameters
of genome evolution and considering either selectionist or
neutralist models (reviewed in [54]). Although selectionist
models related to features such as gene organization
and/or chromatin properties have not been tested, two
observations favor the neutralist model. First, in yeast, the
correlation between DSB levels and GC content is higher
when the GC content at the third base of a codon (GC3) is
used as parameter, rather than GC values as a whole [55].
Second, in mice, the recent translocation of a gene into the
pseudo-autosomal region correlates with an increase in
GC3 from 50 to 73%. This indicates strongly that elevated
GC content is a consequence and not the cause of recombination activity in this case [56]. Two processes have,
therefore, been proposed to explain a neutral effect: either
a mutation bias or a gene-conversion bias [55,57,58]. The
hypothesis that recombination contributes to changes in
GC content also needs to account for the observation that
recombination events are highly localized and, therefore,
explain how, for instance, a local gene-conversion bias
could affect the GC composition over large chromosome
domains. Given the time-scale over which these changes
are expected to take place, the validation of such a hypothesis might require understanding the distribution,
evolution and possible movement of recombination sites
in the genome. Indeed, recombination sites raise a paradoxical situation: given the mechanism of DSBR, sequences
on the active chromosome will be replaced by that of the
less active one, which would be expected to lead to the loss
of initiation sites [59]. However, given the complexity of
determinants that influence recombination rates described
above, and that many of these determinants do not reside
in the area where recombination takes place, the preferential loss of sequences on the initiating chromosome
might only marginally affect its recombination activity.
Conclusions
Many aspects of the mechanism of meiotic recombination
have been identified in yeast, where the initiating lesion
is a DSB. Repair by the DSBR pathway leads to gene
conversion and crossing-over, and repair by the SDSA
pathway leads to gene conversion without crossover. The
main lines of this mechanism are thought to be conserved
in mammals. In S. cerevisiae, analysis of DSBs shows that
initiation takes place in open chromatin. Genetic analysis
shows that both gene conversion and crossover cluster
tightly within a few Kb of the DSB. Comparison of genetic
and DSB data in S. pombe indicates a different situation in
which reciprocal exchanges take place far (. 10 kb) away
from initiation. Although DSBs have not yet been identified in mammals, several crossover hot-spot regions are
likely to correspond to preferred initiation sites. In such
regions, crossovers are tightly clustered and follow a
distribution in agreement with the DSBR pathway, similar
to that described in S. cerevisiae. The heterogeneous
distribution of these hot spots creates large regions with
low levels of recombination separated by small intervals
with hot-spot activities and results in up to thousand-fold
variations in recombination rates. At the chromosome
level in both yeast and mammals, a second layer of
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regulation of recombination is apparent from the presence
of large domains with either high or low levels of recombination. One of the proposed consequences of heterogeneous distribution of recombination, which is supported
by data from yeast and mice, is the increase in GC content
in regions with high levels of recombination. These interpretations provide new ideas and predictions for mechanistic aspects of meiotic recombination.
Acknowledgements
I thank F. Baudat, J. Buard, L. Duret and M. Lichten for critical reading of
the manuscript, and the Centre National de la Recherche Scientifique, the
Commissariat à l’Energie Atomique and the Association pour la Recherche
contre le Cancer for supporting my work.
References
1 Szostak, J.W. et al. (1983) The double-strand-break repair model for
recombination. Cell 33, 25 – 35
2 Pâques, F. and Haber, J.E. (1999) Multiple pathways of recombination
induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63, 349 – 404
3 Belmaaza, A. and Chartrand, P. (1994) One-sided invasion events in
homologous recombination at double-strand breaks. Mutat. Res. 314,
199 – 208
4 Buard, J. et al. (2000) Meiotic recombination and flanking marker
exchange at the highly unstable human minisatellite CEB1 (D2S90).
Am. J. Hum. Genet. 67, 333 – 344
5 Allers, T. and Lichten, M. (2001) Differential timing and control of
noncrossover and crossover recombination during meiosis. Cell 106,
47 – 57
6 Hunter, N. and Kleckner, N. (2001) The single-end invasion: an
asymmetric intermediate at the double-strand break to doubleholliday junction transition of meiotic recombination. PG. Cell 106,
59 – 70
7 Keeney, S. (2001) Mechanism and control of meiotic recombination
initiation. Curr. Top. Dev. Biol. 52, 1 – 53
8 Blat, Y. and Kleckner, N. (1999) Cohesins bind to preferential sites
along yeast chromosome III, with differential regulation along arms
versus the centric region. Cell 98, 249 – 259
9 Blat, Y. et al. (2002) Physical and Functional Interactions among Basic
Chromosome Organizational Features Govern Early Steps of Meiotic
Chiasma Formation. Cell 111, 791– 802
10 Baudat, F. and Nicolas, A. (1997) Clustering of meiotic double-strand
breaks on yeast chromosome III. Proc. Natl. Acad. Sci. U. S. A. 94,
5213– 5218
11 Gerton, J.L. et al. (2000) Inaugural article: global mapping of meiotic
recombination hotspots and coldspots in the yeast Saccharomyces
cerevisiae. Proc. Natl. Acad. Sci. U. S. A. 97, 11383– 11390
12 Nicolas, A. (1998) Relationship between transcription and initiation
of meiotic recombination: toward chromatin accessibility. Proc. Natl.
Acad. Sci. U. S. A. 95, 87 – 89
13 Petes, T.D. (2001) Meiotic recombination hot spots and cold spots.
Nat. Rev. Genet. 2, 360 – 369
14 Davis, L. and Smith, G.R. (2001) Meiotic recombination and chromosome segregation in Schizosaccharomyces pombe. Proc. Natl. Acad.
Sci. U. S. A. 98, 8395 – 8402
15 Pecina, A. et al. (2002) Targeted stimulation of meiotic recombination.
Cell 111, 173– 184
16 Hilliker, A.J. et al. (1994) Meiotic gene conversion tract length
distribution within the rosy locus of Drosophila melanogaster. Genetics
137, 1019– 1026
17 Porter, S.E. et al. (1993) Genetic evidence that the meiotic recombination hotspot at the HIS4 locus of Saccharomyces cerevisiae does not
represent a site for a symmetrically processed double-strand break.
Genetics 134, 5 – 19
18 Schultes, N.P. and Szostak, J.W. (1990) Decreasing gradients of gene
conversion on both sides of the initiation site for meiotic recombination
at the ARG4 locus in yeast. Genetics 126, 813– 822
19 Grimm, C. et al. (1994) M26 recombinational hotspot and physical
conversion tract analysis in the ade6 gene of Schizosaccharomyces
pombe. Genetics 136, 41– 51
Review
TRENDS in Genetics Vol.19 No.9 September 2003
20 Bowring, F.J. and Catcheside, D.E. (1998) Analysis of conversion tracts
associated with recombination events at the am locus of Neurospora
crassa. Curr. Genet. 34, 43 – 49
21 Yeadon, P.J. and Catcheside, D.E. (1998) Long, interrupted conversion
tracts initiated by cog in Neurospora crassa. Genetics 148, 113 – 122
22 Langin, T. et al. (1988) Reciprocal exchanges instigated by large
heterologies in the b2 gene of ascobolus are not associated with long
adjacent hybrid DNA stretches. Genetics 119, 329 – 336
23 Symington, L.S. and Petes, T.D. (1988) Expansions and contractions of
the genetic map relative to the physical map of yeast chromosome III.
Mol. Cell. Biol. 8, 595 – 604
24 Symington, L.S. et al. (1991) Genetic analysis of a meiotic recombination hotspot on chromosome III of Saccharomyces cerevisiae.
Genetics 128, 717 – 727
25 Wu, T-C. and Lichten, M. (1994) Meiosis-induced double-strand break
sites determined by yeast chromatin structure. Science 263, 515– 518
26 Borts, R.H. and Haber, J.E. (1987) Meiotic recombination in yeast:
alteration by multiple heterozygosities. Science 237, 1459– 1465
27 Borts, R.H. and Haber, J.E. (1989) Length and distribution of meiotic
gene conversion tracts and crossovers in Saccharomyces cerevisiae.
Genetics 123, 69 – 80
28 Young, J.A. et al. (2002) Meiotic recombination remote from prominent
DNA break sites in S. pombe. Mol. Cell 9, 253– 263
29 Smith, G.R. (2001) Homologous recombination near and far from DNA
breaks: alternative roles and contrasting views. Annu. Rev. Genet. 35,
243 – 274
30 Jeffreys, A.J. et al. (2000) High resolution analysis of haplotype
diversity and meiotic crossover in the human TAP2 recombination
hotspot. Hum. Mol. Genet. 9, 725– 733
31 Isobe, T. et al. (2002) Molecular characterization of the Pb recombination hotspot in the mouse major Histocompatibility Complex class II
region. Genomics 80, 229 – 235
32 Jeffreys, A.J. et al. (1998) High-resolution mapping of crossovers
in human sperm defines a minisatellite-associated recombination
hotspot. Mol. Cell 2, 267 – 273
33 Jeffreys, A.J. et al. (2001) Intensely punctate meiotic recombination in
the class II region of the major histocompatibility complex. Nat. Genet.
29, 217 – 222
34 Jeffreys, A.J. and Neumann, R. (2002) Reciprocal crossover asymmetry and meiotic drive in a human recombination hot spot. Nat.
Genet. 31, 267 – 271
35 Guillon, H. and De Massy, B. (2002) An initiation site for meiotic
crossing-over and gene conversion in the mouse. Nat. Genet. 32,
296 – 299
36 Borde, V. et al. (1999) Use of a recombination reporter insert to define
meiotic recombination domains on chromosome III of Saccharomyces
cerevisiae. Mol. Cell. Biol. 19, 4832– 4842
37 Cullen, M. et al. (2002) High-resolution patterns of meiotic recombination across the human major histocompatibility complex. Am.
J. Hum. Genet. 71, 759 – 776
38 Kong, A. et al. (2002) A high-resolution recombination map of the
human genome. Nat. Genet. 31, 241 – 247
39 Nachman, M.W. and Churchill, G.A. (1996) Heterogeneity in rates of
recombination across the mouse genome. Genetics 142, 537 – 548
40 Broman, K.W. et al. (1998) Comprehensive human genetic maps:
individual and sex-specific variation in recombination. Am. J. Hum.
Genet. 63, 861 – 869
41 Yu, K. and Feingold, E. (2001) Estimating the frequency distribution of
crossovers during meiosis from recombination data. Biometrics 57,
427 – 434
42 Lawrie, N.M. et al. (1995) Chiasma frequency, distribution and
interference maps of mouse autosomes. Chromosoma 104, 308 – 314
43 Barlow, A.L. and Hulten, M.A. (1998) Crossing over analysis at
pachytene in man. Eur. J. Hum. Genet. 6, 350 – 358
44 Petes, T.D. and Merker, J.D. (2002) Context dependence of meiotic
recombination hotspots in yeast. The relationship between recombination activity of a reporter construct and base composition. Genetics
162, 2049 – 2052
45 Eisenbarth, I. et al. (2000) An isochore transition in the NF1 gene
region coincides with a switch in the extent of linkage disequilibrium.
Am. J. Hum. Genet. 67, 873 – 880
46 Eisenbarth, I. et al. (2001) Long-range sequence composition mirrors
http://tigs.trends.com
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
521
linkage disequilibrium pattern in a 1.13 Mb region of human
chromosome 22. Hum. Mol. Genet. 10, 2833– 2839
Phillips, M.S. et al. (2003) Chromosome-wide distribution of haplotype
blocks and the role of recombination hot spots. Nat. Genet. 33, 382 – 387
Yu, A. et al. (2001) Comparison of human genetic and sequence-based
physical maps. Nature 409, 951 – 953
Fullerton, S.M. et al. (2001) Local rates of recombination are positively
correlated with GC content in the human genome. Mol. Biol. Evol. 18,
1139– 1142
Plug, A.W. et al. (1996) Presynaptic association of Rad51 protein with
selected sites in meiotic chromatin. Proc. Natl. Acad. Sci. U. S. A. 93,
5920– 5924
Bernardi, G. (2000) Isochores and the evolutionary genomics of
vertebrates. Gene 241, 3 – 17
Tenzen, T. et al. (1997) Precise switching of DNA replication timing in
the GC content transition area in the human major histocompatibility
complex. Mol. Cell. Biol. 17, 4043 – 4050
Jabbari, K. and Bernardi, G. (1998) CpG doublets, CpG islands and Alu
repeats in long human DNA sequences from different isochore
families. Gene 224, 123 – 127
Eyre-Walker, A. and Hurst, L.D. (2001) The evolution of isochores.
Nat. Rev. Genet. 2, 549 – 555
Birdsell, J.A. (2002) Integrating genomics, bioinformatics, and
classical genetics to study the effects of recombination on genome
evolution. Mol. Biol. Evol. 19, 1181– 1197
Perry, J. and Ashworth, A. (1999) Evolutionary rate of a gene affected
by chromosomal position. Curr. Biol. 9, 987– 989
Wolfe, K.H. et al. (1989) Mutation rates differ among regions of the
mammalian genome. Nature 337, 283 – 285
Galtier, N. et al. (2001) GC-content evolution in mammalian genomes:
the biased gene conversion hypothesis. Genetics 159, 907 – 911
Boulton, A. et al. (1997) The hotspot conversion paradox and the
evolution of meiotic recombination. Proc. Natl. Acad. Sci. U. S. A. 94,
8058– 8063
Nicolas, A. et al. (1989) An initiation site for meiotic gene conversion in
the yeast Saccharomyces cerevisiae. Nature 338, 35 – 39
Malone, R.E. et al. (1992) A meiotic gene conversion gradient opposite
to the direction of transcription. Nature 359, 154 – 155
White, M.A. et al. (1992) A promoter deletion reduces the rate of
mitotic, but not meiotic, recombination at the HIS4 locus in yeast.
Curr. Genet. 21, 109 – 116
Gyapay, G. et al. (1994) The 1993-94 Genethon human genetic linkage
map. Nat. Genet. 7, 246 – 339
Blake, J.A. et al. (2000) The Mouse Genome Database (MGD):
expanding genetic and genomic resources for the laboratory mouse.
The Mouse Genome Database Group. Nucleic Acids Res. 28, 108 – 111
Smith, R.A. et al. (1998) Recombination breakpoints in the human
beta-globin gene cluster. Blood 92, 4415– 4421
Schneider, J.A. et al. (2002) Direct measurement of the male
recombination fraction in the human beta- globin hot spot. Hum.
Mol. Genet. 11, 207 – 215
Chakravarti, A. et al. (1984) Nonuniform recombination within the
human beta-globin gene cluster. Am. J. Hum. Genet. 36, 1239– 1258
Yip, S.P. et al. (1999) Mapping recombination hotspots in human
phosphoglucomutase (PGM1). Hum. Mol. Genet. 8, 1699– 1706
Han, L.L. et al. (2000) Unequal exchange at the charcot-marie-tooth
disease type 1A recombination hot-spot is not elevated above the
genome average rate. Hum. Mol. Genet. 9, 1881 – 1889
May, C.A. et al. (2002) Crossover clustering and rapid decay of linkage
disequilibrium in the Xp/Yp pseudoautosomal gene SHOX. Nat. Genet.
31, 272 – 275
Khambata, S. et al. (1996) Ea recombinational hot spot in the mouse
major histocompatibility complex maps to the fourth intron of the Ea
gene. Genome Res. 6, 195 – 201
Kobori, J.A. et al. (1986) Molecular analysis of the hotspot of
recombination in the murine major histocompatibility complex.
Science 234, 173– 179
Zimmerer, E.J. and Passmore, H.C. (1991) Structural and genetic
properties of the Eb recombinational hotspot in the mouse. Immunogenetics 33, 132 – 140
Shiroishi, T. et al. (1990) Recombinational hotspot specific to female
meiosis in the mouse major histocompatibility complex. Immunogenetics 31, 79 – 88
522
Review
TRENDS in Genetics Vol.19 No.9 September 2003
75 Shiroishi, T. et al. (1991) Genetic control of sex-dependent meiotic
recombination in the major histocompatibility complex of the mouse.
EMBO J. 10, 681 – 686
76 Snoek, M. et al. (1998) Molecular analysis of the major MHC
recombinational hot spot located within the G7c gene of the murine
class III region that is involved in disease susceptibility. J. Immunol.
160, 266 – 272
77 Sun, H. et al. (1991) Extensive 3(-overhanging, single-stranded DNA
associated with the meiosis-specific double-strand breaks at the arg4
recombination initiation site. Cell 64, 1155– 1161
78 Collins, I. and Newlon, C.S. (1994) Meiosis-specific formation of joint
DNA molecules containing sequences from homologous chromosomes.
Cell 76, 65 – 75
79 Schwacha, A. and Kleckner, N. (1994) Identification of joint molecules
that form frequently between homologs but rarely between sister
chromatids during yeast meiosis. Cell 76, 51 – 63
80 Schwacha, A. and Kleckner, N. (1995) Identification of double
Holliday junctions as intermediates in meiotic recombination. Cell
83, 783 – 791
81 Allers, T. and Lichten, M. (2001) Intermediates of yeast meiotic
recombination contain heteroduplex dna. Mol. Cell 8, 225 – 231
82 Alani, E. et al. (1994) Interaction between mismatch repair and genetic
recombination in Saccharomyces cerevisiae. Genetics 137, 19– 39
83 Cohen, P.E. and Pollard, J.W. (2001) Regulation of meiotic recombination and prophase I progression in mammals. BioEssays 23,
996– 1009
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