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Where the crossovers are: recombination distributions in mammals
Author: A. Kauppi
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"REVIEWS In most sexually reproducing organisms, crossover recombination events provide physical connections between homologous chromosomes. These connec- tions work in conjunction with sister-chromatid cohe- sion to ensure proper chromosome segregation during the first meiotic division 1,2 .The formation of crossovers also has the effect of breaking up HAPLOTYPES,therefore increasing genetic diversity from one generation to the next. Because of this effect, meiotic recombination exerts a profound influence on genome diversity and evolution. Studies in many organisms demonstrate that recombination events are more likely to occur in some parts of the genome than in others. Understanding this nonrandom distribution has important implications for explaining genome plasticity and for our ability to map disease-related loci. In the past few years, our understanding of the distri- bution of recombination events in humans has drasti- cally changed. Recent studies strongly support the view that most recombination events occur at highly localized hot spots, whereas the bulk of the DNA is ?cold?. In this review, we discuss low-resolution experiments that first documented the existence of recombination hot spots in humans and mice, and review more recent high- resolution studies that reveal the highly localized clus- tering of recombination events. Because the emerging pattern in mammals seems similar to that documented in budding yeast (Saccharomyces cerevisiae), we review molecular details in this model organism that are infor- mative for understanding the mechanisms that dictate recombination distributions in mammals. We then dis- cuss the implications that this highly punctuated recom- bination distribution has for understanding genome architecture. Several experimental approaches have been used to characterize the frequency and spatial distribution of meiotic recombination events. All show that recombi- nation in mammals is nonrandomly distributed, at vari- ous levels of spatial resolution (BOX 1).The classic tool in gene mapping is to examine parent-to-offspring trans- mission of alleles at several loci. This so-called pedigree approach has identified localized genomic regions, at the 1?10-kb scale, in which recombination events seem to cluster and that historically were referred to as recom- bination hot spots. However, this approach to measur- ing meiotic recombination rates is limited by difficulties in detecting low-frequency events and by the variable density of polymorphic markers that are used to map crossover positions. Although recombination events are clearly distributed nonrandomly across the genome, it has been difficult to pinpoint precisely where they occur. This issue is important for understanding the WHERE THE CROSSOVERS ARE: RECOMBINATION DISTRIBUTIONS IN MAMMALS Liisa Kauppi* ? ,Alec J. Jeffreys* and Scott Keeney ? Until recently, recombination studies in humans and mice had identified only a few anecdotal examples of crossover hot spots. Recently, the pace of discovery has accelerated. In every genomic segment that has been examined at sufficiently high resolution, recombination events have a punctate recombination distribution: they are clustered within small (1?2-kb) regions that are surrounded by large stretches of recombinationally suppressed DNA. Here, we review progress in understanding the distribution of mammalian recombination events, tie mammalian results together with informative studies in budding yeast and discuss the consequences of these findings for genome diversity and evolution. HAPLOTYPE A combination of alleles at different loci that is transmitted together from one generation to the next. NATURE REVIEWS | GENETICS VOLUME 5 | JUNE 2004 | 413 *Department of Genetics, University of Leicester, Leicester LE1 7RH, UK. ? Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, NewYork, New York 10021, USA. Correspondence to L.K., A.J.J. or S.K. e-mails: kauppil@mskcc.org; ajj@le.ac.uk; keeneys@mskcc.org doi:10.1038/nrg1346 CENTIMORGAN (cM). A measure of genetic distance. 1 cM corresponds to a 1% frequency of recombinant progeny. CHIASMA (pl. chiasmata). A cytologically visible physical connection between homologous chromosomes that corresponds to the position of a meiotic crossover. LINKAGE DISEQUILIBRIUM (LD). A measure of whether alleles at two loci coexist in a population in a nonrandom fashion. Alleles that are in LD are found together on the same haplotype more often than would be expected by chance. MINISATELLITES Regions of DNA in which repeat units of 6?100 bp are arranged in tandem arrays that are 0.5?30 kb in length. 414 | JUNE 2004 | VOLUME 5 www.nature.com/reviews/genetics REVIEWS course, the obvious drawback of sperm PCR techniques is that they can only be used to analyse meiotic crossovers in males. Family data therefore remain essential for analysing female recombination rates in humans. Analysis of flow-sorted single sperm allowed the localization of a hot spot to an 11-kb region in the human ?-globin locus 8 and the demonstration of regional het- erogeneity in crossover rates along the human major histocompatibility complex (MHC) 9 .However,fur- ther narrowing of hot spots in both studies was lim- ited by the number of crossovers that were recovered and the density of markers that were analysed. Other studies using the batch PCR approach have been able to identify and characterize several crossover hot spots in humans and mice at even higher resolution. High-resolution analysis of human crossover hot spots. The first hot spot to be characterized by sperm DNA analysis lies next to the MINISATELLITE MS32 on chromo- some 1 (REF. 7).This hot spot is highly localized (it is ~1.5-kb wide) and is centred 200 bp away from the minisatellite, with crossovers extending into the array of repeat DNA. The peak crossover rate is 50 times the male genome average. High-resolution LD mapping across 210 kb of the MHC class II region revealed several localized regions of LD breakdown (see FIG. 2b for an example). Allele-specific PCR analyses of sperm DNA identified 6 localized crossover hot spots (some of them in clusters that span molecular mechanism behind nonrandom recombina- tion distributions because our perspective is strongly influenced by the spatial resolution of the methods that are used to detect recombination events 3 . Characterizing mammalian hot spots Progress in this field has recently accelerated owing to two technical advances: the development of efficient re- sequencing methods for SNP discovery, which have greatly increased marker density 4 , and, more impor- tantly, the advent of single-molecule methods that allow recombinant DNA molecules to be recovered directly from sperm DNA. There are two variations on the latter theme. One relies on the PCR amplification of haploid genomes of individual flow-sorted sperm 5 (reviewed in REF. 6). The other approach uses allele-specific PCR to amplify recombinant DNA molecules directly from large batches of sperm DNA 7 (FIG. 1); this approach allows many thousands of meiotic products from a sin- gle individual to be screened, and provides the means to detect and quantify even very low-frequency recombi- nation events (10 ?6 or less per sperm). Sperm analysis can be coupled to linkage disequilibrium (LD) surveys (BOX 2; FIG. 2), using the latter as a guide to the location of putative hot spots. Crossover-breakpoint mapping and the resolution of hot spots is limited only by the density of markers within the target region; for humans, resolution to less than 0.5 kb is usually possible by choosing individuals with appropriate genotypes. Of Box 1 | Crossover distributions in mammals ? an historical perspective Genetic versus physical distances Crossover activity in mammals is usually expressed in CENTIMORGANS (cM) per megabase pair (Mb). Comparisons of genetic distances with physical distances reveal, at the megabase level, long DNA domains of elevated or suppressed recombination that alternate along the length of each chromosome (see, for example, REFS 61,91). Because estimates of genetic distance are based on the number of offspring observed with recombinant genotypes, the resolution of genetic maps is typically limited to 0.1?1 Mb, so little is known about how crossovers are distributed within ?hot? and ?cold? domains. Linkage maps also reveal that females have a higher recombination rate than males, with the female:male genetic-map-length ratio being approximately 1.6:1 (REFS 40,92).Female:male ratios are typically highest around centromeres and lowest near telomeres 40,60,92,93 . Cytological studies Crossover distributions can also be examined cytologically by observing CHIASMATA or through immunofluorescent detection of chromosome-associated protein complexes that are involved in recombination. These cytological approaches reveal that most chromosomes in humans and mice undergo one or two crossovers per chromosome or chromosome arm in each meiosis, with species-specific and sex-specific variations in position along chromosomes 88,94?100 .However, these studies do not provide a high-resolution view of recombination distributions because of low sample sizes and microscopic resolution. Pedigree and linkage-disequilibrium analysis The average crossover frequency is 0.5 cM Mb ?1 in mice 101 and 1.1 cM Mb ?1 in humans 40 . This means that, within a 1-kb interval, crossovers in humans will occur on average in only 1 gamete in 90,000, and therefore only the most intense hot spots will be detected by pedigree analysis. An alternative approach is to use LINKAGE DISEQUILIBRIUM (LD) measures of the association of alleles at separate loci to estimate the amount of historical recombination that has occurred between the loci (BOX 2),and therefore to infer the presence and location of crossover hot spots 102 .Pedigree approaches, often guided by LD analysis, have identified several putative crossover hot spots in the human genome, although the number of crossovers that are detected in each region are low (for example, the ?-globin gene region 103,104 ,the PGM1 gene 105 ,the ADA gene 106 ,the LRP5 gene 107 and three regions of the major histocompatibility complex (MHC) 87,108 ). In the mouse MHC, 4 regions that are active in recombination have been defined at a resolution of <10 kb 109?116 .In fact, the first direct evidence for crossover hot spots in any mammal came from mouse MHC studies 113 .Also, the elegent work of Shiroishi and co-workers on the mouse Lmp2 hot spot highlights the potential importance of cis-acting factors in mammalian recombination 114,117 . NATURE REVIEWS | GENETICS VOLUME 5 | JUNE 2004 | 415 REVIEWS similarities with one another. Interestingly, some hot spots are located in repeat elements (such as Alu ele- ments and LINEs), which raises the issue of how ectopic recombination events are prevented in the human genome. Thuriaux pointed out that recombination might be associated with genes, because in various organisms the overall amount of recombination corre- lates with gene content rather than the physical size of the genome 16 .However, there does not seem to be a strict relationship of this sort for mammalian hot spots. The human and mouse MHC hot spots and the SHOX hot spot are all located at or near genes (within ~8 kb), but, as the MHC is a gene-rich region and SHOX was pre-selected for analysis, this association is not necessar- ily meaningful. Only one of these recombination hot less than 10 kb) in these regions of LD breakdown 10,11 (FIG. 3).All of these hot spots show clustering of crossovers within a narrow (1?2-kb) region, and are surrounded by DNA sequences that are virtually inert for recombination (FIG. 3).Peak recombination activities at these MHC hot spots vary markedly, from 0.3 cM Mb ?1 to 110 cM Mb ?1 .The peak activity at the weakest hot spot is actually below the genome average fre- quency, but the hot spot is nevertheless evident given the extremely low recombination rate in surrounding sequences, which is estimated to be approximately 0.05 cM Mb ?1 (REF. 11).For this reason, it is useful to define hot spots as localized sites of recombination rather than as sites that show recombination frequencies above the genome average. With this concept of a hot spot, it is worth noting that there is no need to invoke the exis- tence of specific chromosomal sites with opposite prop- erties ? that is,?cold spots?. Indeed, most of the DNA sequences in the genome are cold. The 2.6-Mb pseudoautosomal pairing region (PAR1) at the subtelomeric region of Xp/Yp is a site of obligatory crossover in male meiosis. High-resolution LD mapping around the SHOX gene in PAR1 showed no evidence of clear LD block structure 12 ,which is consistent with the high rate of crossing over in males (20-fold above the genome average) 13 .However,a sperm-crossover assay targeted to 1 of the regions of free association in the SHOX gene revealed a highly localized (2-kb-wide) hot spot, with peak crossover activities of 140?370 cM Mb ?1 in the 3 men tested 12 . High-resolution analysis of mouse recombination hot spots. Recently, two mouse MHC hot spots have been subjected to similar crossover analyses in sperm 14,15 . The Lmp2 (now re-named Psmb9) gene region con- tains a hot spot that is similar in width (1.8 kb) to all human hot spots and with an intense peak activity of 1,300 cM Mb ?1 (REF. 14).At the Eb hot spot, which is again of similar width, the influence of haplotype on male meiotic recombination has been analysed in detail 15 .Analysis of sperm crossovers from mice that are heterozygous for different haplotypes showed large variation in recombination rates that correlated inversely with sequence divergence between haplotypes in and near the hot spot, although the position of the hot spot remained unchanged. Properties of crossover hot spots The studies described above reveal several features of recombination hot spots. Crossovers at all mammalian hot spots identified so far cluster within small (1?2- kb) regions (FIG. 3b); these common widths imply that there are common mechanisms of recombination ini- tiation and resolution at different hot spots. Crossover breakpoints are distributed symmetrically (that is, they have a ?normal distribution?; see FIG. 3c) within these narrow regions. Peak recombination rates at human hot spots vary from below the genome average at 1 hot spot in the MHC to 370-fold above the genome average at the SHOX hot spot. Hot spots are located in different genomic environments and share no obvious sequence LD BLOCK A DNA segment within which markers are in significant linkage disequilibrium with each other, which implies that there is low recombination activity within the block. a LD analysis b Crossover analysis Target region SNP discovery LD mapping Hot-spot candidate region Allele-specific PCR Crossover-breakpoint mapping Figure 1 | The PCR approach for recovering crossover molecules directly from sperm DNA. a | The first step is SNP discovery (SNPs are shown as beige circles) and linkage-disequilibrium (LD) mapping, which allow the identification of LD BLOCKS (orange boxes) and regions where LD breaks down (gap between orange boxes). Such regions of LD breakdown are putative recombination hot spots. b | For putative recombination-hot-spot regions, two rounds of allele-specific PCR are carried out on batches of sperm DNA from an individual with multiple SNP heterozygosities (red and green circles). Allele-specific primers are shown as red and green arrowheads. Recombinant sperm DNA molecules are selectively amplified, and the location of crossover breakpoints can be subsequently mapped by typing internal SNPs. 416 | JUNE 2004 | VOLUME 5 www.nature.com/reviews/genetics REVIEWS The how and why of nonrandom distributions The existence of localized recombination hot spots in mammals raises the question of how this punctate crossover distribution arises. A large body of work, pri- marily in budding yeast, provides a direct insight into the molecular mechanisms that are behind hot-spot activity. The DNA events of meiotic recombination. The meiotic recombination pathway in budding yeast has at its heart the formation and subsequent repair of double-strand breaks (DSBs; reviewed in REFS 21?23; see FIG. 4). The repair of any given meiotic DSB results in either the rec- iprocal exchange of the chromosome arms that flank the break (a crossover) or no exchange of flanking arms (a non-crossover or parental configuration). It is highly probable that DSBs initiate meiotic recombination in mammals and other organisms as well, not least because the central player in DSB formation, Spo11, is structurally and functionally conserved 24,25 . Crossover distributions reflect double-strand-break dis- tributions. In budding yeast, the single most important determinant of the crossover distribution is the distribu- tion of the DSBs that initiate meiotic recombination 17,26 . As for crossover distributions in humans (BOX 1), DSB distributions in budding yeast show many levels of spa- tial organization (FIG. 5).There are hot (DSB-rich) and cold (DSB-poor) domains that span large portions of chromosomes (in the order of 100 kb) 17,27,28 .Within these large domains, DSBs form preferentially at local- ized hot spots 26,29,30 . Finally, within any given hot spot, spots (the weakest MHC hot spot) lies in a promoter region, in contrast to budding yeast in which pro- moter sequences are preferential sites for hot spots 17 . Furthermore, some human MHC genes probably do not have adjacent hot spots 11 , and the MS32 hot spot lies in the middle of a 77-kb non-coding DNA segment, again arguing against a 1:1 correlation between genes and hot spots. Crossover hot spots are also gene-conversion hot spots. There is evidence that recombination hot spots in mice and humans are sites of initiation and resolution of both crossovers and non-crossover gene conver- sions 7,14,18 .At Lmp2 in the mouse MHC, conversions that involve a single chosen marker in the hot spot revealed short conversion tracts of <540 bp 14 .More recently, an ENRICHMENT-BASED ASSAY 19 has been developed to capture conversion events that involve even single SNP sites 18 .Analysis of three human crossover hot spots showed that they are also extremely localized conversion hot spots, with peak crossover and conversion activity mapping to the same location at the centre of the hot spot. The number of non-crossover conversion events dominated the number of crossovers by a 4:1 to 15:1 ratio, conversion tracts were short (<300 bp on average) and initiation events were inferred to cluster within a small zone of ~400 bp 18 .The existence of initiating lesions in hot spots has been confirmed by Qin et al. 20 , who developed a PCR-based approach to reveal the presence of meiosis-specific strand breaks with 3?-OH ends in the mouse Ea recombination hot spot. GENE CONVERSION The non-reciprocal transfer of information between homologous DNA sequences as a consequence of heteroduplex formation during recombination, which is followed by repair of mismatches in the heteroduplex. ADMIXTURE The mixing of two genetically differentiated populations. ENRICHMENT-BASED ASSAY An allele-specific hybridization method for enriching DNA molecules that carry certain allelic combinations from bulk genomic DNA. Box 2 | D? as a measure of linkage disequilibrium Linkage disequilibrium (LD) is a population-based parameter and reflects recombination in all of the generations since the rise of the individual polymorphic alleles that are under study. So, LD analyses do not suffer from the same limitations as do pedigree analyses with respect to detecting low-frequency events. However, patterns of LD can be influenced by the age of crossover events, GENE CONVERSION,selective advantages or disadvantages of single or linked alleles, and demographic history, such as founder effects and ADMIXTURE 84,118,119 . In principle, if strong LD is found between two loci, little if any historical recombination has occurred between them. Conversely, little or no LD implies that there has been historical recombination activity. There are various LD measures for characterizing the statistical association between alleles at different loci (see, for example, REF. 120). A simple measure of LD is D ij = x ij ? p i q j ,where x ij is the observed frequency of haplotype A i B j ,p i and q j are the frequencies of alleles A i and B j at loci A and B, respectively, and the expected frequency of gamete A i B j is p i q j assuming no statistical association between the alleles. The range of this measure is a function of the allele frequencies, and is therefore not ideal. Consider 2 SNPs in which SNP1 carries alleles C and G (at a population frequency of 0.90 and 0.10, respectively), and SNP2 carries alleles A and T (at a population frequency of 0.55 and 0.45, respectively). There are then 4 possible haplotypes for these 2 SNPs (CA, CT, GA and GT); assuming no statistical association, we expect to see these haplotypes at frequencies of 0.495, 0.405, 0.055 and 0.045, respectively. If the observed haplotype frequencies in the population are 0.450, 0.450, 0.100 and 0, then D CA = 0.450 ? 0.495 = ?0.045. The normalized measure D? (REF. 121) is perhaps the most widely used, as its range is the same for all allele frequencies. It does tend to be over-inflated when sample size is small, however 122 .D? is defined as follows: D? ij = D ij /D max ,where D max = min[p i q j ,(1?p i )(1?q j )] when D ij <0, or D max = min[p i (1?q j ), (1?p i )q j ] when D ij >0. For the above example of the CA haplotype, D max = 0.045, which is the lesser value of 0.9�0.55 = 0.495 and (1?0.9)(1?0.55) = 0.045. Therefore, D? CA = D CA /D CA max = ?0.045/0.045 = ?1. D? can range from ?1 to 1; whether the sign is positive or negative depends on the arbitrary labeling of alleles, and therefore, the absolute value |D?| is mostly used. If |D?| = 1, this is referred to as complete LD, and occurs if 3 haplotypes at most are present in the population. The presence of three haplotypes can be explained by mutation alone, and therefore, there is no evidence for historical recombination. If |D?| = 0, this is referred to as no LD, or free association, and implies that there has been extensive recombination between the markers, or (in theory) recurrent mutation. LD plots can be generated to visualize the extent of LD (see FIG. 2). The statistical significance of each LD value can also be incorporated into the plots. NATURE REVIEWS | GENETICS VOLUME 5 | JUNE 2004 | 417 REVIEWS DSB levels and insertions into hot regions give high DSB levels 41,42 .This phenomenon supports a model in which higher-order structures and/or chromosome dynamics contribute to the control of the frequency of recombina- tion-initiation events, but at present the molecular basis of this process is not known. DSB-hot domains in bud- ding yeast correspond to regions with relatively G+C-rich sequences 17,28,43 .Importantly, crossovers in mammals also form preferentially in chromosomal domains that corre- spond to similar G+C-rich ISOCHORES or ?R-BANDS? 40,44,45 (although at the local level, there is no evidence that mammalian hot spots and their immediate environments are unusually G+C rich). It therefore seems probable that hot and cold chromosomal domains are specified by similar processes in humans and in budding yeast. Within these larger domains, DSBs in budding yeast form within localized hot spots. If appropriately analysed by Southern blotting of restriction digests of whole genomic DNA, these hot spots appear as clusters of frequently cleaved sequences, which typically span ~70?250 bp and are surrounded by sequences in which breaks form rarely, if at all 17,31?34 (see FIG. 5c). The punctate distribution of DSBs at this spatial resolution is highly reminiscent of the punctate distribution of the recombi- nation events that were described above for the MHC and other regions in mammals. The similarity of these patterns in turn leads, in a straightforward way, to the idea that mammalian hot spots correspond to the sites at which DSBs form preferentially. specific phosphodiester bonds are cleaved at different frequencies 31?34 .This last level of spatial resolution is dic- tated at least in part by Spo11 itself because certain spo11 missense mutations alter the distribution of cleavage sites within hot spots 35 . Detailed reviews of fungal hot spots have been pro- vided elsewhere 3,36?39 ;here we highlight those features that will probably provide insight into the molecular mechanisms that are behind nonrandom crossover dis- tributions in mammals. It should be kept in mind that recombination per unit of physical distance in budding yeast (0.37 cM kb ?1 for 12�10 6 bp total) is much higher than in humans (0.001 cM kb ?1 for ~3�10 9 bp total) 38,40 . Therefore, individual hot spots in budding yeast are much more active in recombination: a 1?10% crossover frequency is not unusual for budding-yeast hot spots 3 , whereas a 0.3% crossover frequency is the ?record? in humans so far (at the SHOX hot spot 12 ). The presence of DSB-hot and DSB-cold chromoso- mal domains has been amply documented across the budding-yeast genome 17,27,28 . On chromosome 3, for example, regions near the telomeres show few or no prominent DSBs. There are also few DSBs near the cen- tromere, whereas interstitial regions in each of the chro- mosome arms show a high frequency of DSBs (FIG. 5a). This bias in the distribution of DSBs represents a large- scale position effect, because a recombination reporter that is placed at different positions takes on the proper- ties of its location: insertions into cold regions give low LIKELIHOOD RATIO The relative likelihood of obtaining observed experimental data under two different models. ISOCHORE A region of genomic DNA sequence in which G+C compositions are relatively uniform. R-BANDS A chromosome banding pattern that is produced with various staining procedures; it is reciprocal to the pattern that is produced by Giemsa staining (G-Bands). 10 kb LD blocks LD breakdown LD blocks LD breakdown 123 546 2 3 4 5 6 a SNPs: a hypothetical genomic region 1 DMB b SNPs: an MHC region <20 20?100 100?1,000 1,000?10,000 >10,000 Upper left: LR versus free association 1 >0.8 0.6?0.8 0.4?0.6 0.2?0.4 0?0.2 Lower right: D? Figure 2 | Graphical presentation of linkage-disequilibrium data. a | Linkage disequilibrium (LD) in a hypothetical genomic region. In this example, there are 6 SNPs, numbered 1?6 (labelled below and to the left of the plot). LD is calculated for each pair of SNPs from genotypes of a panel of individuals. Each pairwise comparison is plotted as a rectangle centred on each SNP, and extending half way to adjacent markers. The |D?| values are plotted by colour code (see key) below the diagonal. The statistical significance of each |D?| value can be deduced from the LIKELIHOOD RATIO (LR) versus free association, which is shown above the diagonal. In this example, |D?| = 1 between markers 1 and 2, indicating complete LD. This value is supported by a high (>10,000) LR. By contrast, the |D?| value between markers 3 and 5 is 0.4?0.6, but this value is accompanied by poor odds (LR versus free association <20). A region of LD breakdown is apparent between markers 2 and 4 (arrow). b | LD in a portion of the human major histocompatibility complex (MHC). Patterns of LD near the DMB gene are shown (data from REF. 11). Only SNPs with a minor allele frequency ?0.15 have been included. LD blocks are identified visually and are shown as orange boxes. There are two regions of LD breakdown (vertical arrows). Adapted with permission from REF. 11 � (2001) Macmillan Magazines Ltd. 418 | JUNE 2004 | VOLUME 5 www.nature.com/reviews/genetics REVIEWS important for targeting the DSB machinery, either by inducing a local chromatin structure that is permissive for DSB formation, or by recruiting the recombination- initiation machinery by direct protein?protein interac- tions 48,53?55 .Petes and colleagues have argued that there are in fact three classes of recombination hot spot: those that require transcription factors for targeting recombi- nation complexes, those that are transcription-factor- independent because they are always in a permissive state for DSB formation and those that are hot because of a high G+C content, without the necessity of being in an open chromatin configuration 28,38,55 . Other sources of nonrandomness. If the number of early forming chromosome-associated complexes of the strand-exchange proteins Rad51 and Dmc1 is used to estimate the total number of recombination events, it is apparent that meiotic cells make a large excess of DSBs relative to the number of crossovers that eventually form (see, for example, REFS 56,57). Because every DSB must be repaired, it follows that the excess breaks must be processed to give a non-crossover configuration. The cytological data in mammals indicate that non- crossovers outnumber crossovers by approximately 10:1 The factors that determine whether a given DNA sequence will be a DSB hot spot in budding yeast are not completely understood, but some general rules have emerged. One important determinant is the chro- matin structure. Essentially, all known DSB hot spots are nuclease-hypersensitive in both mitotic and meiotic chromatin, and the results of several experiments indicate that an open chromatin configuration is necessary for DSB formation 26,29,30,46,47 .But chromatin structure cannot be the sole arbiter because not all nuclease-hypersensitive sites are DSB hot spots and there is no correlation between the degree of nuclease hypersensitivity and the frequency of DSB formation 26,29,30,41,42,47 .Most naturally occurring hot spots in budding yeast (but not in mam- mals) lie in promoter regions 17,36 .However, transcription per se is not required for DSB formation, because deletion of the TATA box at HIS4 markedly diminishes transcrip- tion without affecting the recombination frequency 48 , and because several artificial hot spots have been created that seem not to be transcriptionally active 41,49?52 .The tendency of DSBs to occur in promoter regions might reflect the fact that promoters tend to have an open chro- matin configuration. Alternatively, sequence-specific binding proteins (such as transcription factors) might be D N A D M A D M B P S M B 9 P S M B 8 T A P 1 T A P 2 R IN G 3 3 mcM 3 mcM 4 mcM ? 050100 150 200 0.01 0.1 1 10 100 1,000 Hot spot DNA3 cM Mb ?1 cM Mb ?1 1 6 56 5 1 1 150 100 50 0 cM Mb ?1 200 100 0 a bc kb 32 33 34 35 36 37 kb 32 33 34 35 36 37 kb Figure 3 | Highly localized recombination activity in the human major histocompatibility complex class II region. The presence of peaks in crossover activity, which are embedded in ?cold? DNA, are reminiscent of recombination patterns in maize (for example, the 140-kb interval between the anthocyanin1 and shrunken2 genes, REF. 126), and might be typical of higher eukaryotes with complex genomes. a | Genes in the region are shown as boxes above the plot. The peaks in recombination activity represent the six crossover hot spots characterized by sperm analyses (see main text). The approximate recombination rate for inter-hot-spot DNA segments is given in millicentiMorgans (mcM, cMx10 ?3 ). Mean male recombination rate (0.81 cM Mb ?1 , REF. 40) is shown as a dashed line. The most active crossover hot spot, DNA3 (shown in more detail in b and c), is indicated with a dashed box. b | Within the DNA3 hot spot, crossovers cluster into a narrow (less than 2-kb) region. A total of 70 crossovers were recovered from 57,000 sperm DNA molecules from 1 man. Orange bars indicate the crossover activity in cM Mb ?1 in each heterozygous SNP interval (SNPs shown as vertical bars above the plot). The number of crossovers that map to each interval is shown above the bars. c | Underlying morphology and crossover activity of the DNA3 hot spot (based on the least-squares best-fit curve for the crossover data, assuming a normal distribution of crossover breakpoints). Peak crossover activity was determined from the mean rate in the six men tested. Adapted with permission from REF. 11 � (2001) Macmillan Magazines Ltd. NATURE REVIEWS | GENETICS VOLUME 5 | JUNE 2004 | 419 REVIEWS on the Y chromosome). Another possible example of region-specific bias in the crossover/non-crossover ratio is the tendency of crossovers to be relatively telomere-proximal in human males 60,61 .Alterations in the crossover/non-crossover ratio might also explain why some organisms show changes in the frequency of chias- mata that are formed adjacent to heterozygous configura- tions of large-scale chromosome rearrangements such as translocations or inversions (see, for example, REFS 62,63). Finally, crossover frequencies are suppressed when the recombining DNA molecules contain extensive sequence differences, as has been demonstrated in interspecific crosses in bacteria and budding yeast 64,65 .Although it is not understood precisely which stage in the recombina- tion process is affected by sequence heterology, the involvement of the cellular mismatch-repair machinery in inhibiting recombination makes it probable that this effect occurs during or after strand exchange. Why is the distribution of recombination events nonran- dom? Because nonrandom crossover distributions are so widespread in evolution, the question arises as to what purpose is served by controlling the spatial organi- zation of recombination events. One possibility is that the cell must balance two opposing needs: recombina- tion is necessary to ensure proper chromosome segre- gation at the first meiotic division, but it must also be reined in to minimize the breaking up of favourable linkage groups and to foster genome stability. Perhaps controlling the spatial distribution of crossovers provides a way to achieve this balance. However, comparison of mouse and human MHC class II hot-spot distributions shows that although gene order in the mouse and human MHC is conserved, crossover hot-spot locations do not seem to be conserved (FIG. 6).This would indicate first, that hot-spot location is not dictated by ?linkage groups? of particular genes, or at least that the require- ment for genes contained within a haplotype is differ- ent in the two species. Second, the comparison shows that recombination hot spots are relatively transient, with a life span that is at least shorter than the time since mouse?human divergence. Evidence from LD studies that the human ?-globin hot spot is not active in rhesus macaques (or if it is, it has not left its imprint on haplotype diversity) is consistent with this 66 . Another possibility is that restrictions on crossover position facilitate the optimal mechanical/biochemical function of chiasmata in chromosome segregation. In the light of this, it is noteworthy that errors in chromosome segregation are correlated with unusual crossover posi- tions in many cases of maternally derived trisomy 21 (REFS 67,68).What is ?optimal? might vary significantly from organism to organism and even between sexes in the same species or among chromosomes in the same cell. Finally, deliberately inflicting DSBs on its own genome is a dangerous game for the organism to play: a single unrepaired break will probably be lethal, yet hun- dreds of breaks are made in each meiotic cell. Perhaps restrictions on the spatial organization of DSB formation promote the efficient repair of DSBs by ensuring that breaks form in regions of the genome that are conducive (REF. 58).This estimate applies to the genome as a whole; however, the bias in the outcome of DSB repair at any given hot spot or within a given chromosomal domain could differ substantially from this genome average. In principle, then, regional variation in crossover frequen- cies could reflect not only variation in DSB frequency, but also variation in how probable it is that a DSB will give rise to a crossover instead of a non-crossover outcome. There are several potential examples of this idea. In budding yeast, the average crossover/non-crossover ratio is ~1:2 (measured as the fraction of gene conver- sions that are associated with the crossing over of flank- ing markers), but ratios at different loci have been reported to range from ~1:4 to ~2:1 (REF. 59).In mam- mals, the most striking possible example of this phe- nomenon is the obligate crossover within the PAR1 region of the XY-chromosome pair. As judged by immunostaining for recombination proteins, DSB for- mation is distributed along the lengths of the X and Y chromosomes, forming in both the PAR and the non- homologous chromosome-arm regions 58 .The forma- tion of a crossover specifically within PAR1 in every male meiosis might therefore reflect a regional bias towards a crossover outcome rather than (or in addition to) an increase in DSB frequency relative to other parts of the genome (presumably, the DSBs outside the PAR are repaired by recombination between sister chro- matids or between repeated sequences that are dispersed HOLLIDAY JUNCTION A point at which the strands of two double-stranded DNA molecules exchange partners, which occurs as an intermediate in crossing-over. Dmc1, Rad51 and other proteins Spo11 3? 3? Crossover Non-crossover Figure 4 | The meiotic recombination pathway in budding yeast. Physical analysis of DNA isolated from meiotic cell cultures has defined the important steps along the recombination pathway, starting with the initiating double-strand breaks (DSBs) 49,127,128 . DSB formation is catalysed by Spo11, which seems to act through a topoisomerase-like reaction to generate a transient, covalent protein?DNA intermediate 129,130 . Spo11 is then removed from the DNA and the 5?-strand termini are nucleolytically resected to yield variable-length, 3?-single-strand tails 131,132 . In a series of reactions that are dependent on budding-yeast homologues of bacterial RecA 132,133 , these tails undergo strand invasion of intact homologous duplexes, ultimately giving rise, in at least some cases, to double HOLLIDAY-JUNCTION intermediates, followed by mature recombinant products. 420 | JUNE 2004 | VOLUME 5 www.nature.com/reviews/genetics REVIEWS similar to that documented across large portions of the genome in budding yeast. So, it seems probable that this pattern will turn out to be the rule rather than the exception for mammals as well, with most recombina- tion events occurring within such localized hot spots in every meiosis. If the average spacing between hot spots or hot-spot clusters within the human MHC class II region (~65 kb on average) is representative of spacing in the 3.2�10 9 -bp genome, then there should be ~50,000 recombination hot spots. Such a punctate crossover distribution would have several consequences for genome structure and evolution. LD blocks in the human genome. Recently, it has emerged that a large part of the human genome consists of so- called LD, or haplotype, blocks 11,69?72 .Within each block, polymorphic markers are in association with each other, with the net effect that there is a limited repertoire of haplotypes (reviewed in REF. 73). This block structure and limited haplotype diversity provides an important tool to simplify the search for genetic determinants of common disease (BOX 3). On the basis of the precedent set by the MHC (see above), it has been suggested that the block structure in the rest of the genome is mainly a consequence of highly localized recombination hot spots being separated by recombinationally ?cold? DNA (the cold sequences would therefore make up the blocks themselves) 69,71 .It is impor- tant to note, however, that recombination distributions probably do not explain all aspects of haplotype-block structure. Genetic drift can generate haplotype blocks in the absence of recombination hot spots, as shown by various simulation studies 72,74?76 .There is also some experimental evidence that highly active recombination hot spots do not necessarily break down LD fully (A.J.J., unpublished observations), and that not all regions of LD breakdown contain (sperm) recombination hot spots (L.K. and A.J.J., manuscript in preparation). Recently, hot-spot locations and crossover activity have been estimated using COALESCENT APPROACHES, and exam- ples so far seem to agree remarkably well with sperm- crossover data 77,78 .Such approaches might prove more useful for predicting hot spots than LD/haplotype structure alone. In addition to contributions to the block-like haplo- type structure, the occurrence of punctate recombina- tion distributions imposes important considerations for the evolutionary dynamics of haplotype diversity. First, most gene-conversion events should only be able to cre- ate novel allelic combinations at a localized level within hot spots, and therefore should have little, if any, effect on haplotypes that are contained within long LD blocks (there is, however, some evidence that rare gene conver- sion outside hot spots might contribute to haplotype diversification within LD blocks 79 ). Second, the exis- tence of LD blocks results in the correlated behaviour of multiple SNPs. Specifically, within a recombinationally suppressed region of DNA, the fixation or extinction of alleles will not occur independently; instead, loss of a haplotype from a population will result in the loss of all variants that are restricted to that haplotype. to proper assembly of DSB-repair complexes, and by ensuring the appropriate coordination of recombina- tion with the formation of higher-order chromosome structures. Effects on genome structure and evolution In every case in which mammalian recombination events have been analysed at sufficiently high resolution, crossovers and non-crossover conversions cluster within small regions that are surrounded by large stretches of recombinationally suppressed DNA. Because such high- resolution data are so far available for only a few (and perhaps atypical) loci in mice and humans, extrapolat- ing this pattern to the genome as a whole warrants cau- tion. Nevertheless, this highly punctuated distribution is COALESCENT APPROACHES A means of investigating the shared genealogical history of genes. A genealogy is constructed backwards in time starting with the present-day sample. Lineages coalesce when they have a common ancestor. Pulse-field Conventional agarose Acrylamide 317 kb 362 bp 304 bp 246 bp Hour 0 2 7 ~215 kb ~80 kb 6.2 kb 3.7 kb 2.5 kb 9.6 kb Mabc Figure 5 | Nonrandom distribution of double-strand breaks in budding yeast, revealed by different resolutions of gel analysis. a | The double-strand break (DSB) distribution across the entire length of a budding yeast chromosome (chromosome 3) is revealed by electrophoresis followed by Southern blotting and indirect end-labelling with a probe directed to sequences near the end of the chromosome. Samples were prepared from a synchronous cell culture at various times after induction of meiosis. Immediately before entry into meiosis (0 h), only full-length chromosomes are seen. As the cells progress into meiosis, faster migrating species are observed, corresponding to chromosomes that have suffered one or more DSBs. Approximate sizes of prominent bands are indicated. b | At higher resolution, a single band on a pulse-field gel can be resolved into several discrete regions of DSB formation (hot spots) that are separated by regions with few or no DSBs. In this example, genomic DNA was prepared 7 h into meiosis, digested with a restriction enzyme, separated and analysed by Southern blotting and indirect end-labelling with a probe derived from sequences in the ARE1 gene. The full-length parental band is 9.6 kb; each of the DSB bands maps to the promoter of a gene in this region. Approximate sizes of some of the bands are indicated. c | At even higher resolution, DSBs within a hot spot occur at particular phosphodiester bonds that are distributed nonrandomly across ~150 bp. In this example, digested genomic DNA was analysed by Southern blotting after electrophoresis. Sizes of molecular weight standards are shown (lane M). For all three panels, samples were prepared from strains carrying a mutation (rad50S) in which DSBs form but are not processed further. Adapted with permission from REF. 17 � (1997) National Academy of Science and REF. 33 � (1995) Macmillan Magazines Ltd. NATURE REVIEWS | GENETICS VOLUME 5 | JUNE 2004 | 421 REVIEWS over-transmitted to crossover molecules, which results in different crossover-breakpoint locations for the reciprocal crossover configurations. Such over-transmission is evi- dent at a human MHC hot spot, in which DSBs are pref- erentially initiated on the chromosome that carries the A allele of a G/A SNP near the centre of the hot spot, lead- ing to an approximately 87:13 over-transmission of the non-initiating G allele to crossover progeny 82 .The result is weak but significant MEIOTIC DRIVE in favour of the fixa- tion of the non-initiating G allele. Crossover asymmetry (disparity) has also been found at other hot spots in humans and mice 7,15 ,which is consistent with the ?hot- spot conversion paradox? 83 whereby hot spots should self- destruct through meiotic drive consistently in favour of recombination-suppressing variants. Meiotic drive. The DSB-repair model for meiotic recom- bination 80 (FIG. 4) predicts that the chromatid on which the DSB is made is the recipient of information. This pre- diction is borne out in studies in budding yeast (see, for example, REF. 81). If DSB formation at a particular hot spot has an equal chance of occurring on either homo- logue, then within a population of recombinant mole- cules, the crossover breakpoints should map to the same locations for the 2 reciprocal crossover configurations, and alleles at any polymorphic sequences within the hot spot itself should be transmitted in a 1:1 ratio (see REF. 82 for a detailed discussion). On the other hand, if one homologue is preferentially used for DSB formation, crossover asymmetry is observed: alleles near the hot spot centre that are on the non-initiating chromosome are MEIOTIC DRIVE Distortion of meiotic inheritance such that one allele at a heterozygous site is recovered in greater than half of the gametes (as opposed to the expected 50:50 Mendelian segregation). Pseudogenes DPB2 COL11A2 DP A2 RPL32L RING13 HLA-Z RING14 IPP2 RING8 RING9 DQB2 DQA2 DQB3 COX3-L GLN-tRNA DRB2 Genes DPB1 DP A1 DNA RING3 DMA DMB PSMB9 T AP1 PSMB8 T AP2 DOB DQB1 DQA1 DRB1 DRB3 DRA BTL I I DRB9 Pb Pa Eb2 Btlii-like Pseudogenes Oa Frg1 Ma Mb Mb1 Lmp2 T ap1 Lmp7 T ap2 Ob(Ab2) Ab1 Aa Eb1 Ea Btlii Genes Crossover hot spots Human 100 kb Mouse Crossover hot spots ? ? Figure 6 | Comparison of crossover hot-spot locations in the human and mouse major histocompatibility complex class II region. Genes are shown as red boxes, and pseudogenes as blue boxes. Orthologous genes are connected with lines (adapted from REF. 134). Known hot spots (REFS 10,11,14,15,109?114,116, 135 and L. K. and A.J.J., manuscript in preparation) are indicated with arrows. Mouse hot spots were, however, identified using pedigrees and it is probable that there are further weaker hot spots in this region of the mouse major histocompatibility complex (MHC). Regions in the human MHC that have not yet been targeted for high-resolution linkage disequilibrium and/or sperm-crossover analyses are marked with a thick green line. Box 3 | Using haplotype-block structure in genetic mapping Gray et al. 123 , and a little later Johnson et al. 124 ,pointed out that for a non-recombining haplotype block, a lower density of (SNP) markers is sufficient for disease-mapping purposes. Such SNPs were called ?haplotype-tag SNPs? (htSNPs) 124 . An htSNP could capture the information of its haplotype block, and therefore cut down the cost and labour of having to analyse a much higher number of SNPs. Simplifying the search for genetic determinants of common disease immediately sparked huge interest. htSNPs should reduce the problem of disease mapping to two steps: finding haplotype blocks, and then, within the patient cohort, finding the haplotype in the block with the strongest association to disease. The HapMap Project,launched in 2001, aims to map haplotype-block structure in DNA samples from Nigeria, Japan, China and the United States, and to define htSNPs for each block 125 .Although this will yield no direct information on the cause of haplotype blocks, it will generate data on how long and how ?universal? haplotype blocks are on average. Some words of caution are needed. The definition of haplotype blocks is always dependent on marker density ? it should be kept in mind that for some genomic regions or in certain populations, high-frequency SNPs, or indeed clear block boundaries, might simply not exist. As yet, it is unclear what statistical thresholds are appropriate to define block boundaries. Depending on the (arbitrary) stringency of block definition, a large LD block might be broken into shorter sub-blocks, each of lower diversity. It is also possible that an aetiological variant lies inside a recombination hot spot and would be impossible to identify using a haplotype-tagging strategy. 422 | JUNE 2004 | VOLUME 5 www.nature.com/reviews/genetics REVIEWS domains and at localized hot spots (see, for example, REFS 7,9,15,40,82,88,89). When considering recombination in the context of selection, it is remarkable how, in the human MHC at least, the distribution of sperm crossovers agrees with patterns of LD, which is a population-based measure of recombination. The former involves direct detec- tion of recombinants that have not been subjected to natural selection (apart from successfully completing meiosis), whereas the latter consists of a collection of different haplotypes that have succeeded in surviving in the population in the face of selective forces. This indi- cates that in this segment of the MHC, selection has had little effect on the length of haplotype blocks and supports the idea that recombination hot spots, rather than population-genetic factors, are key players in shaping diversity. Of course, it is possible that the loca- tion of the hot spots is dictated by selection in the first place. In the light of this, it would be interesting to see if hot-spot locations are conserved between humans and evolutionarily much closer species, such as the great apes. Future challenges include identifying and character- izing more examples of mammalian hot spots to begin to understand the factors that govern their distribution. The HapMap Project (BOX 3) should reveal how general the block-like patterns of LD in humans are and how long, on average, these blocks are. Once a large number of regions of LD breakdown have been identified, they could in theory be systematically targeted for sperm- crossover assays. The drawback of sperm PCR methods lies in the laborious fine-tuning of the assays, which makes them almost impossible to scale up. Alternative approaches are therefore necessary to analyse crossovers on the whole-genome scale; haplotyping individual DNA molecules across putative crossover hot spots using single-molecule imaging or the PCR COLONY METHOD 90 might be one way to do this. Nucleotide diversity. An inverse correlation between nucleotide diversity and LD has been noted 84 , and there seems to be a strong positive correlation between nucleotide variability and recombination rate at the megabase level 85 . One way to account for these observations is to postulate that recombination might be mutagenic. Indeed, homologous recombina- tion that is induced by a DSB in mitotically growing budding-yeast cells does seem to be mutagenic 86 , although it is not known whether this is true of mei- otic recombination as well. Alternatively, the localized nature of meiotic recombination events predicts that SNPs within and near a recombination hot spot might be able to escape correlated shifts in allele frequencies that occur within the LD blocks that flank the hot spot. Specifically, alleles within hot spots could avoid rapid correlated extinctions by being constantly reshuffled onto different haplotypes. This idea is sup- ported by the relatively stable levels of nucleotide diversity that are seen in hot spots, but not LD blocks, across populations 79 . Conclusions For the MHC, LD data and limited numbers of famil- ial crossovers 87 are in good agreement with sperm- crossover data, which indicates that the same hot spots also function in females but at different levels of activity. So, the most intense MHC hot spot (DNA3) shows similar levels of activity in males and females, whereas a hot spot in the TAP2 gene seems to be some 30-fold ?hotter? in women 10 .Therefore, the same genomic locations might be potential recombination hot spots in all individuals, but there could be some mechanism that sets the crossover activity to a different level in the two sexes or for dif- ferent haplotypes. Variation in recombination rates between individuals has been noted at the genome- wide level, as well as for relatively large chromosomal PCR COLONY METHOD A method in which individual DNA molecules are immobilized in a matrix and then subjected to PCR amplification in situ.For (haploid) sperm DNA, polymorphic sites can be typed within amplification products to yield haplotypes of chromosomal regions, allowing, in principle, the identification of recombinant molecules. 1. Petronczki, M., Siomos, M. F. & Nasmyth, K. 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Online links DATABASES The following terms in this article are linked online to: Entrez: http://www.ncbi.nih.gov/Entrez ADA | ARE1 | HIS4 | Lmp2 | LRP5 | PGM1 | SHOX | Spo11 | TAP2 FURTHER INFORMATION Alec Jeffreys? laboratory: http://www.le.ac.uk/ge/ajj/index.html GermOnline (a web database for information about germ cell biology, including meiosis): http://germonline.unibas.ch International HapMap Project: http://www.hapmap.org Scott Keeney?s laboratory: http://www.ski.edu/keeney Access to this links box is available online. "
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