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Homologous chromosome interactions in meiosis: diversity amidst conservation
Author: Jennifer L. Gerton
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"� 2005 Nature Publishing Group *The Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110, USA. ? University of Kansas Medical Center, Department of Biochemistry and Molecular Biology, 4011 Wahl Hall East, 3901 Rainbow Boulevard, Kansas City, Kansas 66160. � University of Kansas, Department of Molecular Biosciences, 2034 Haworth Hall, 1200 Sunnyside Avenue, Lawrence, Kansas 66045. Correspondence to J.L.G. e-mail: JEG@Stowers- Institute.org doi:10.1038/nrg1614 From the point of view of the genome, meiosis is a matter of matching, locking and moving chromo- somes. These processes are crucial for creating gam- etes that have the correct number of chromosomes. In humans, up to 30% of spontaneous miscarriages are estimated to be the result of chromosome mis- segregation events 1 . The results of only a few misseg- regation events are compatible with human life, and these include Down (trisomy 21), Turner (mono- somic for X) and Klinefelter (XXY male) syndromes. In most organisms, chromosome missegregation events that occur during meiosis result in inviabil- ity. Although many of the mechanisms and proteins that participate in meiotic pairing and segregation are evolutionarily conserved, there are definite spe- cies-to-species differences in the main mechanisms used to achieve homologue segregation. One of the main mechanisms by which homolo- gous chromosomes are locked together involves crossing over, which is the result of recombination events that are initiated by double-strand breaks (DSBs). It is vitally important that in the face of the high frequency of recombination that occurs during meiosis, the genome is faithfully maintained. This is accomplished by adding specialized layers of regula- tion on top of the normal homologous recombination machinery. However, there is also evidence to indi- cate that at least some organisms, such as yeast and flies, are not entirely dependent on recombination for locking their homologous chromosomes together and instead can use other mechanisms to distribute their chromosomes 2 . In this review, we focus on meiosis-specific mech- anisms that drive interaction between homologous chromosomes and how these vary between organ- isms. We begin with an overview of meiotic events, followed by a description and comparison of DSB- independent and dependent matching and locking mechanisms. In particular, we focus on one example of diversity amidst the conserved need to match and lock homologous chromosomes: the function of the Mnd1?Hop2 protein complex. We conclude by highlighting some of the future challenges in the field. HOMOLOGOUS CHROMOSOME INTERACTIONS IN MEIOSIS: DIVERSITY AMIDST CONSERVATION Jennifer L. Gerton* ? and R. Scott Hawley* � Abstract | Proper chromosome segregation is crucial for preventing fertility problems, birth defects and cancer. During mitotic cell divisions, sister chromatids separate from each other to opposite poles, resulting in two daughter cells that each have a complete copy of the genome. Meiosis poses a special problem in which homologous chromosomes must first pair and then separate at the first meiotic division before sister chromatids separate at the second meiotic division. So, chromosome interactions between homologues are a unique feature of meiosis and are essential for proper chromosome segregation. Pairing and locking together of homologous chromosomes involves recombination interactions in some cases, but not in others. Although all organisms must match and lock homologous chromosomes to maintain genome integrity throughout meiosis, recent results indicate that the underlying mechanisms vary in different organisms. NATURE REVIEWS | GENETICS VOLUME 6 | JUNE 2005 | 477 REVIEWS nrg0605-hawle.indd 477nrg0605-hawle.indd 477 17/5/05 5:20:45 pm17/5/05 5:20:45 pm � 2005 Nature Publishing Group Leptotene Zygotene Pachytene Early Mid Late DSB Nascent interaction Stable strand invasion Strand extension by DNA synthesis Double Holliday junction Increasing degrees of homologue interaction Alignment Axes formation; pairing Bridge formation Synapsis; SC formation SC stabilization HOLLIDAY JUNCTION A point at which the strands of two dsDNA molecules exchange partners, which occurs as an intermediate during genetic recombination. COHESIN A multi-protein complex that maintains tight association of sister chromatids. CHROMOSOME TERRITORY A domain of the nucleus occupied by a pair of homologous chromosomes. CHIASMA (Pl. chiasmata.) A cytologically visible physical connection between homologous chromosomes that corresponds to the position of a meiotic cross-over. Overview of meiotic events Meiosis is a special set of cell divisions that produces gametes in organisms that reproduce sexually. Cells enter meiosis in much the same way as they enter mitosis ? having replicated their chromosomes. However, in cells that undergo meiosis, this replica- tion might involve further specialized features that facilitate later phases of meiosis. For example, repli- cated sister chromatids in both mitosis and meiosis are held together by COHESIN; all eukaryotes studied so far have meiosis-specific versions of this complex. One of the unique aspects of meiosis is that homologous chromosomes must be identified and locked together in a way that allows them to segregate from each other at the first meiotic division (meiosis I). Homologous chromosomes in most organisms interact through recombination to produce at least one cross-over per chromosome so that they can segregate properly at the first nuclear division. The segregation of homologues at the first meiotic division is followed by the dissolution of chromosome cohesion and the segregation of sister chromatids at the second meiotic division (meiosis II). As there is not an intervening S phase, the result is a gamete that contains half the number of chromosomes of the starting cell. What are the mechanisms used to identify homo- logues and lock them together? Homologue identifica- tion is perhaps the most mysterious aspect of meiosis. It might involve any or all of the following, depending on the species: interactions between DNA duplexes, interactions between DNA and proteins at specialized pairing centres, interactions between centromeres and centromeric heterochromatin, interactions between telomeres, and CHROMOSOME TERRITORIES. The evidence for each of these is discussed below. Homologues are held together during much of the meiotic prophase by a proteinaceous structure known as the synaptonemal complex 3 . A more permanent linkage occurs as a result of recombination and the associated crossing over of DNA that generates CHIASMATA, the cyto- logical manifestations of cross-overs. The frequency of such reciprocal recombination events is not uniform; rather, in some regions of the euchromatin, levels of recombination are much higher than in others (het- erochromatic regions do not undergo crossing over). Moreover, when analysed at the DNA-sequence level, even those exchanges that occur in recombination-rich regions of euchromatin tend to occur at certain places in the genome, which are termed recombination hotspots, where double-strand breaks occur at a high frequency 4 . In most organisms, homologues are held together by both cross-overs and the synaptonemal complex, although a few organisms have only the syn- aptonemal complex, such as Bombyx mori females, or only cross-overs, such as Schizosaccharomyces pombe. Besides homologue matching and locking mecha- nisms, there are other mechanisms that are required for correct homologue segregation at the first meiotic divi- sion, which are the processes that facilitate the correct orientation of homologous centromeres on the meiotic spindle. However, for the purpose of this review we concentrate on mechanisms that promote interactions between homologous chromosomes. Matching of homologues During the early stages of meiotic prophase, pairs of homologous chromosomes are matched by mecha- nisms that are incompletely understood. The process of homologue interaction can be conceptualized as increasing degrees of physical association between homologues over time, culminating in close, stable homologue juxtaposition (FIG. 1). The term alignment usually refers to bringing homologous chromosomes into rough apposition along their entire length. Pairing refers to the intimate association of homologues. Synapsis is often used to refer to chromosomes that are connected by the synaptonemal complex that comes to lie between homologues and connects them along their entire length. Homologue matching in some species depends on the formation of a set of meiosis- specific DSBs, which are induced by proteins that are Figure 1 | Homologue interactions during meiosis. During chromosome pairing that is independent of double-strand break (DSB) formation (alignment), regions of local distortion might allow homology to be sensed. During DSB-dependent homologue interactions (pairing and nascent interactions), 3? single-stranded regions engage in interactions with the homologous chromosome. During synapsis and synaptonemal complex (SC) formation, 3? ssDNA ends stably invade the homologue. The synaptonemal complex, a proteinaceous structure, forms between homologous chromosomes. During this phase, the invading strand is extended by DNA synthesis. Once the strand is recaptured, a double HOLLIDAY JUNCTION forms. Adapted, with permission, from REF. 9 ? (2001) Elsevier Science. 478 | JUNE 2005 | VOLUME 6 www.nature.com/reviews/genetics REVIEWS nrg0605-hawle.indd 478nrg0605-hawle.indd 478 17/5/05 5:20:49 pm17/5/05 5:20:49 pm � 2005 Nature Publishing Group homologous to the Saccharomyces cerevisiae meiosis- specific sporulation protein (Spo11) and that initiate the recombination process; in other species, matching can occur by DSB-independent means. Although DSB- independent processes seem to be sufficient for homo- logue matching in flies and worms, DSB-dependent processes seem to be essential for accurate homologue interactions in yeast, Arabidopsis thaliana, mice and humans. DSB-independent matching of homologues DSB-independent pairing processes seem to be wide- spread, and at least in some cases, these interactions precede DSB-dependent pairing. In S. cerevisiae, for example, DSB-independent pairing is detected at the beginning of meiosis and then becomes undetectable during the meiotic S phase, but pairing is quickly re-established in a DSB-independent manner after S phase 5 . The molecular nature of these DSB-inde- pendent pairing interactions is unclear, but might simply involve interactions that are based on homology between two DNA duplexes. It has been argued that these types of ?kissing? interaction would be transient and unstable, and would not require an active process to dissociate them 6 . Their stabilization could occur through several sites of interaction along the length of each chromosome or through stabilizing proteins. Live imaging of chromosome movements in S. pombe revealed an initial steep increase in homologous pairing at the beginning of meiosis in wild-type cells and in a mutant lacking DSBs (a mutant for rec12; the S. pombe homologue of S. cerevisiae SPO11). This pairing was not detected in a meu13 (the S. pombe homologue of S. cerevisiae HOP2) mutant, arguing that meu13 might have a role in early DSB-independent pairing interac- tions 7 . DSB formation soon follows, however, and at least in S. cerevisiae, mice and A. thaliana, formation of the synaptonemal complex between homologues seems to depend on DSBs and is concomitant with stable strand invasion ? when a single-stranded 3? DNA end from one homologue stably invades the homologous sequence on the other homologue 8?12 . In some organisms, DSB-independent pairing processes seem to be sufficient for synapsis and syn- aptonemal complex formation. Caenorhabditis elegans chromosomes enter meiosis unpaired and then undergo a rapid alignment. This alignment requires neither the initiation of recombination nor the function of proteins that will later facilitate synapsis 13 . Homologue alignment and pairing also occur normally in the complete absence of DSBs in both sexes of Drosophila melanogaster 14,15 . In D. melanogaster mutants in which recombination levels are at least 100-fold below normal, the synaptonemal complex assembles perfectly normally between homo- logues 15 . Similarly, in the fungus Coprinus cinereus, a significant amount of homologue pairing occurs even when meiotic DSBs are absent 16 . The lack of a require- ment for DSBs to initiate synapsis in these organisms probably reflects the ability of flies and worms to use other DSB-independent means to mediate homologue recognition, such as somatic pairing and the use of pairing centres. Somatic pairing as a basis for homologue pairing. One reason that DSBs might not be necessary for homologue pairing in D. melanogaster is that the chro- mosomes are already aligned when meiosis begins. Mitotic chromosomes in somatic and germline cells in D. melanogaster and other Dipterans have been known for almost a century to have high levels of homologue pairing 17?19 . Hexaploid wheat also has somatically paired homologues 20 . Using chromosomes that were tagged Table 1 | Methods to monitor homologue interactions Method Use Advantage Disadvantage One-dimensional gel followed by Southern blot To monitor DSBs, heteroduplex DNA and recombination products Easy to quantify Only monitors one genomic locus Two-dimensional gel followed by Southern blot To monitor the formation of single-end invasions and joint molecules Easy to quantify Only monitors one genomic locus Tetrad dissection To monitor recombination outcomes and spore viability Can monitor multiple chromosome intervals Monitoring recombination requires live meiotic products FISH; GFP?lac repressor To monitor pairing of particular sites on homologues (for example, centromeres, telomeres or individual sites) An established technique Might require spreading of chromosomes; timing can be crucial; might only be able to monitor one locus at a time Immunostaining To monitor foci formation of specific proteins (for example, antibodies against Dmc1 or Rad51 identify recombination sites) An established technique Might require spreading of chromosomes; timing can be crucial Live chromosome imaging Homologue pairing; chromosome movements Can monitor all chromosomes at all times Technically challenging FISH, fluorescence in situ hybridization. NATURE REVIEWS | GENETICS VOLUME 6 | JUNE 2005 | 479 REVIEWS nrg0605-hawle.indd 479nrg0605-hawle.indd 479 17/5/05 5:20:51 pm17/5/05 5:20:51 pm � 2005 Nature Publishing Group bcdef 1�m a Telomeres Centromeres Spindle pole body HETEROSOMES Homologous chromosomes that are not identical in appearance; for example, the sex chromosomes. with the GFP?lac repressor protein at different posi- tions uniF6AETABLE 1uniF6AF, Vazquez et al. followed the behaviour of meiotic chromosomes during male D. melanogaster meiosis 21 . Because meiosis in D. melanogaster males occurs in the absence of recombination or a recogniz- able synaptonemal complex, this is a good system in which to study homologue matching and segregation in the complete absence of recombination. The results indicated that the pairing events observed in male meiosis I were simply the result of the continuation of previous somatic pairings, and not the result of a meio- sis-specific mechanism 21 . The authors proposed that heterochromatic associations, including centromere associations (see below) or chromatid entanglement, might be responsible for the maintenance of homo- logue association. They also demonstrated that the sequestering of paired homologues into chromosome territories might have an active role in ensuring the specificity of meiotic segregation (see below). Pairing centres. There is mounting evidence for the existence of specific cis-acting sequences that aid homo- logue identification in worms and flies. In C. elegans, specific cis-acting regions, or homologue-recognition regions (HRRs), have been identified at the ends of each of the 6 chromosomes. Only a piece of a chromosome that retains the HRR is capable of recombining with its intact homologue 22,23 . These sites might be required for the initiation or maintenance of pairing or synapsis 24 . How they function is still rather mysterious. A different type of pairing centre is exemplified by homologous regions that are used for pairing of oth- erwise non-homologous chromosomes. Perhaps the best example of this type of pairing site is the rDNA region of the X and Y chromosomes that mediates sex- chromosome pairing in D. melanogaster males 25?27 . A pseudoautosomal region on the human X and Y chro- mosomes, PAR1, could also be considered an example of a pairing site or region. Most X and Y chromosomes are non-homologous. The PAR1 region is the single largest region of homology between the X and Y chro- mosomes, and it is therefore the site of obligatory pair- ing and exchange between the HETEROSOMES. The W and Z chromosomes in birds are similarly non-homologous and their pairing and exchange is regulated by a pseu- doautosomal region. The sequencing of the platypus genome has recently revealed that this organism has 10 sex chromosomes, which indicates that these 5 pairs of heterosomes must each have a unique pseudoau- tosomal region, or pairing and recombination centre, to generate XXXXX gametes for females and YYYYY gametes for males 28 . Pairing that involves centromeres and heterochroma- tin. Centromeres and/or pericentric heterochromatin might function as a type of pairing centre in the sense that these cis-acting regions on each chromosome might help homologues to find one another. In an interesting exception to the locking of homologous chromosomes through recombination, homologues that lack cross-overs (non-exchange homologues) in D. melanogaster oocytes are matched and segregated in meiosis in a way that depends on the pairing of the pericentric heterochromatin 29,30 . Other organisms also seem to have mechanisms for distributing chromosomes in the absence of reciprocal exchange, often referred to as distributive mechanisms. In S. cerevisiae, a single chromosome pair that does not undergo exchange segregates correctly in 90% of meioses 31?34 ; this phenomenon also seems to be medi- ated by centromere association. Centromeres of non- exchange chromosomes seem to pair in meiosis and mediate bipolar attachment to the spindle at the first meiotic division 35 . So, centromeres and pericentric het- erochromatin might be special cis-acting regions that can facilitate homologue segregation in the absence of recombination. Pairing that involves telomeres. Cytogenetic studies uniF6AETABLE 1uniF6AF of the first meiotic prophase of many organ- isms have revealed the clustering of telomeres at the nuclear periphery during the leptotene?zygotene transition 36?40 (FIG. 2). Because this configuration, known as the chromosomal bouquet, precedes the initiation of synapsis, the possibility has been raised that this early localization of the telomeres to a small region of the nuclear envelope facilitates the align- ment of homologous chromosomes. Saccharomyces cerevisiae strains that contain a mutation in spo11 (which lack DSBs) or that carry a specific mutation in the DNA repair gene rad50 (the rad50S mutants make DSBs but cannot further process them) form bouquets 41 , demonstrating that in S. cerevisiae, bou- quet formation is independent of recombination and synapsis 38 . In a spo11 mutant in Sordaria macrospora, Figure 2 | Telomere clusters during meiosis in budding yeast. a | A schematic representation of telomere configuration during meiosis in budding yeast. Telomeres are released from the nuclear envelope at the time of DNA replication. Through recombination initiation in the late leptotene, telomeres reattach to the nuclear envelope. At the leptotene?zygotene transition, the telomeres cluster tightly near the spindle pole body in a ?bouquet?. At pachytene, telomeres again disperse around the nuclear envelope. Adapted, with permission, from Nature REF. 44 ? (2005) Macmillan Magazines Ltd. b?f | A confocal image of intact Saccharomyces cerevisiae nuclei that are stained with DAPI (blue; indicates DNA), FITC (green; indicates telomeres) and rhodamine (red; indicates a cosmid of chromosome 11). b | The pre-meiotic nucleus with several peripheral telomere clusters and separate cosmid signals. c | The pre-meiotic nucleus with a similar telomere distribution, but with associated cosmid signals. d | The meiotic nucleus with telomere clusters and separate cosmid signals. e | The meiotic nucleus with clustered telomeres and paired cosmids. f | The late meiotic nucleus, with peripheral clustered telomeres and paired cosmids. Reproduced, with permission, from REF. 38 ? (1999) Company of Biologists Ltd. 480 | JUNE 2005 | VOLUME 6 www.nature.com/reviews/genetics REVIEWS nrg0605-hawle.indd 480nrg0605-hawle.indd 480 17/5/05 5:20:53 pm17/5/05 5:20:53 pm � 2005 Nature Publishing Group Nascent DSB; partner complex Onset of stable strand exchange CO nodule + SC patch ASTRAL MICROTUBULES Microtubules that extend from each pole of the mitotic spindle without attaching to any other visible structure. CHROMOSOME PAINTING Fluorescence in situ hybridization (FISH) to chromosomes using a probe that represents a whole chromosome or part of a chromosome. the bouquet also forms normally, arguing that DSBs are not needed for this type of chromosome organiza- tion 42 . However, spo11 mutants in both S. macrospora and budding yeast cannot exit from this phase of meiosis 38,42 . Exogenous DSBs rescue this delay in S. macrospora, which indicates that exit is mediated by regulatory processes that sense the progression of recombination beyond the DSB stage 42 . Ndj1, a meiosis-specific telomere protein required for bouquet formation, is required for the localization of telomeres to the nuclear periphery during meiotic prophase in budding yeast. In an ndj1 mutant, linear chromosomes (but not ring chromosomes, which lack telomeres) missegregate at a high rate 36,43 . Meiotic divisions and synaptonemal complex formation are delayed 36,43 , as is the processing of DSBs into recom- bination products 44 . Together, these results have been taken to indicate that Ndj1 might facilitate recombi- nation through its effect on meiotic telomere organi- zation. It might do this by kinetically facilitating the homologue search through contributions to coalign- ment, or by reducing the effective volume between homologous sites in the nucleus 45 . During homologue pairing in S. pombe, the nucleus oscillates between the cell poles, a process that is driven by ASTRAL MICROTUBULES. During these oscillations, the telomeres are clustered at the spindle pole body, located at the leading edge of the moving nucleus, and the rest of each chromosome follows behind. This oscillatory nuclear movement during meiotic prophase depends on cytoplasmic dynein 46 . A mutation in the dynein heavy chain gene (dhc1) reduces nuclear movement; conse- quently, homologous centromeric and arm regions fail to associate 47 . This gene is required for chromosome segregation in the presence and absence of recombi- nation 118 . Another mutation, in a gene that encodes a telomere-binding protein and length regulator, taz1, allows association of homologous centromeres, but disrupts the association of arm and telomere-proximal loci 47 . In addition, S. pombe mutants that have disrupted telomere clustering show reduced recombination 48,49 , which indicates that telomere clustering facilitates the homologue alignment that is crucial for recombination. In mice and humans, centromere and telomere move- ments during the early meiotic prophase are associated with the onset of chromosome pairing 50 . Chromosome territories. Cytogenetic studies in several organisms show that different chromo- somes occupy particular domains or territories in the nucleus. The separation of chromosomes into domains might facilitate their pairing by effectively reducing the volume of the nucleus. A deletion of the homologous pairing suppressor locus (ph1b) in rye results in a low rate of chromosome synapsis 51 . Comparison of isogenic lines with and without the ph1 mutant locus demonstrates similar pre-meiotic chromosome arrangement and telomere configura- tions. However, although homologous chromosomes in the wild-type line maintain separate territories in the nucleus, they become intermingled in the ph1b mutant line 51 . Furthermore, although centromere associations occur in the mutant, unlike in the wild type, these associations are non-homologous 52 . In the nucleus of D. melanogaster, spermatocyte chro- mosome territories might also promote the maintenance of meiotic pairing 21 . CHROMOSOME PAINTING in human spermatogonia reveals compacted, largely mutually exclusive chromosome territories 50 , again indicating that localization of homologues to a domain might aid in maintaining alignment and pairing. DSB-dependent matching of homologues In most organisms, the interaction of meiotically induced DNA DSBs with matching sequences on the homologous chromosome brings homologous chromosomes into alignment during the early- to mid-leptotene stage. DSBs are made by the topoi- somerase type II-like protein, Spo11 uniF6AEREFS 53,54uniF6AF. The sites of DSB-dependent homologue interactions can be seen as ~400-nm bridges between chromosome axes 55,56 (FIG. 3). These bridges, which probably contain a DSB that is already engaged in a nascent interaction with its partner DNA, occur in large numbers 9,57,58 . As leptotene proceeds, a small fraction of these bridges seem to mature into structures known as axial associations that connect the paired lateral elements 59 . These axial associations will eventually nucleate the formation of the synaptonemal com- plex (that is, as synapsis-initiation sites) between paired chromosomes. The topic of synaptonemal complex formation is beyond the scope of this review, but has been the subject of other recent reviews (see for example REFS 2,4). The formation of DSBs might be linked to chro- matin structure. It has been shown that DSBs tend to occur in regions of open chromatin, or regions that are hypersensitive to nucleases 60?62 . They are also positively correlated with double promoter regions or head-to- head promoters in S. cerevisiae 63 . Binding of transcrip- tion factors, but not transcription itself, is crucial for hotspot activity at certain locations in the genomes of yeast and mice 64?69 . Certain chromatin states might be specific to meiosis or facilitate recombination. Some might promote DSB formation, whereas others might follow DSB formation to promote resolution of breaks. Recently, it has been shown that ubiquitylation of histone H2B is necessary Figure 3 | Visualization of chromosomal bridges in Allium fistulosum and Allium cepa (plant) meiocytes. The sites of double-strand break (DSB)-dependent homologue interaction can be seen as ~400-nm bridges between chromosome axes 55,56 . These bridges, which probably contain a DSB that is already engaged in a nascent interaction with its partner DNA, occur in large numbers 9,57,58 . Their formation depends on the RecA (recombination protein) homologues that are expressed in this species. In the next phase of homologue interaction, these nascent interactions are converted to stable strand-invasion events. This nucleates the formation of the synaptonemal complex (SC). Reproduced, with permission, from REF. 119 ? (1987) Springer. NATURE REVIEWS | GENETICS VOLUME 6 | JUNE 2005 | 481 REVIEWS nrg0605-hawle.indd 481nrg0605-hawle.indd 481 17/5/05 5:20:55 pm17/5/05 5:20:55 pm � 2005 Nature Publishing Group Spo11 Rad50?Xrs2? Mre11 Rad51?Dmc1 Dmc1 Resolution to CO DSB 5? end resection Homology search Dmc1?Mei5? Sae3-mediated strand invasion End extension End recapture (JM) Dmc1 Rad50?Xrs2?Mre11Spo11 Rad 51 for recruiting and/or stabilizing the DSB-forming machinery that contains Spo11 in budding yeast 70 . HIM-17 (high incidence of males due to increased X-chromosome loss 17), which is required for the cor- rect accumulation of histone H3 methylation at lysine 9 on meiotic prophase chromosomes, is essential for DSB formation but dispensable for homologous synapsis in C. elegans 71 . In mammalian cells, regions that contain DSBs become phosphorylated on the histone variant H2AX (?-H2AX) 72 . Although there is certainly a link between chromatin structure and homologue pairing and recombination, the nature and the hierarchy of these events are just beginning to be explored. The nature of the nascent interactions between a DSB and its partner DNA is unclear. Spo11 makes a DSB using its catalytic tyrosine to attack the phosphodiester backbone of the DNA, creating a covalent bond between Spo11 and the 5? end of the break. The resec- tion of the 5? end of the break requires a protein com- plex that contains the DNA repair proteins, Xrs2, Mre11 and Rad50, exposing a 3? single-stranded tail (FIG. 4). This 3? tail might be involved in paranemic interactions (in which one strand is in close proximity to the dsDNA with which it shares homology) and then increasingly stable plectonemic interactions (in which one strand is wound around the dsDNA with which it shares homology) with homologous sequences before any type of stable strand invasion. The interac- tions between the 3? single-stranded tail and duplex DNA might constitute a DSB-dependent homology search that allows DSB-dependent homologue pair- ing to occur. As these types of interactions occur in several locations along the length of homologous chromosomes, they would probably increase the extent of homologue pairing. Once several of these interactions are established, the chromosome axes might become stably aligned 56 . A homology search is probably mediated at least in part by the Escherichia coli recombination protein RecA homologues Rad51 and Dmc1, which are thought to bind to the 3? single- stranded DNA tail. RecA has been shown to mediate this type of homology recognition 73 . Studies in many organisms have shown that synap- sis depends on DSBs. In yeast, mouse and A. thaliana individuals that carry mutations in the SPO11 homo- logues, the synapsis of homologous chromosomes is much reduced or undetectable 8,12,74 . In the absence of Spo11-induced DSBs, synapsis can be restored if DSBs are induced by other means, as has been shown experi- mentally for mutations in SPO11 or its homologues in S. cerevisiae, C. cinereus and the mouse 12,16,75 . It is most likely that further processing of the DSBs into recom- bination intermediates is also required for correct and efficient synapsis. Analysis of yeast mutants in which the production or processing of DSBs is impaired reveals various synaptonemal complex formation defects 11,76 . Recombination creates a physical association Stable strand invasion, which can be detected by two-dimensional gel electrophoresis 9 uniF6AETABLE 1uniF6AF or can Figure 4 | The molecular mechanism of meiotic crossover recombination. Meiotic recombination begins with a double-strand break (DSB) made by Spo11 (a meiosis-specific sporulation protein) through a transesterification reaction (see main text for details). The 5? ends of this break are resected, leaving stretches of 3? ssDNA. This DNA is probably used in a homology search that involves the RecA (recombination protein) homologues Dmc1 and Rad51. Once the appropriate homologue is identified, Dmc1 begins a crossing-over recombination event by mediating stable invasion of the homologous chromosome using one of the 3? ends. This is followed by DNA synthesis that extends the end of the invading strand, and then by recapture of this strand, which generates a joint molecule (JM) that contains a double Holliday junction. This can then be resolved into a cross-over. The connection between two homologous chromosomes through a double Holliday junction is crucial for accurate homologue segregation at the first meiotic division. 482 | JUNE 2005 | VOLUME 6 www.nature.com/reviews/genetics REVIEWS nrg0605-hawle.indd 482nrg0605-hawle.indd 482 17/5/05 5:20:58 pm17/5/05 5:20:58 pm � 2005 Nature Publishing Group HETERODUPLEX DNA DNA that contains a strand from each homologue. be inferred by the presence of HETERODUPLEX DNA 77,78 , occurs at the beginning of synapsis and synaptonemal complex formation in S. cerevisiae 9 . The appearance of stable strand-invasion events depends on the E. coli RecA homologue Dmc1 uniF6AEREFS 9,79uniF6AF. Both Rad51 and Dmc1 have been shown to mediate strand invasion in vitro 80,81 . Other proteins are required for strand exchange, including Tid1?Rdh52, Rad52, and Rad54 (for a detailed review of the role of Rad52 epistasis group proteins in recombination and DSB repair see REF. 82). The protein complexes Mei5?Sae3 and Mnd1?Hop2 have recently been suggested to assist in meiotic strand invasion; we will concentrate on these newcomers in this review. Stable strand-inva- sion events can mature into the cross-overs and chi- asmata that are needed to lock homologues together for correct segregation. Recombination is biased towards interhomologue recombination by Red1?Hop1. Once DSBs are made, ostensibly they can be resealed, undergo recombina- tion with the sister chromatid, undergo recombination with the homologue or undergo recombination with an ectopic location that probably shares some homology. Each DSB hotspot is likely to have different frequen- cies of each fate, but the most likely fate of a DSB is recombination with a homologue. Budding yeast con- tains genes that seem to specifically channel breaks into interhomologue recombination. Red1 and Hop1 are part of the chromosome axis and Mek1 is a meio- sis-specific serine?threonine kinase 83,84 . Together, these proteins are thought to channel DSBs into a Dmc1- dependent interhomologue-recombination pathway 85 . In their absence, the number of DSBs is reduced, and the remaining DSBs are mainly resolved by intersister recombination 86 . This does not prevent sporulation, but spores have poor viability as they lack the cross- overs necessary to facilitate appropriate homologue segregation. At present, it is not clear whether other organisms have similar proteins that direct DSBs into interhomologue recombination. The meiotic recombinases Rad51 and Dmc1 and Mnd1?Hop2. At least two RecA homologues function during meiosis in S. cereviseae. One is Rad51, which also participates in homologous recombination in mitosis, and some organisms contain multiple para- logues of this protein. The second protein, Dmc1, is a meiosis-specific RecA homologue which is present in some organisms 87 (see below). Both Rad51 and Dmc1 have been shown to mediate strand invasion in vitro 80,81 . Yeast that lack Rad51 can still carry out some recombination and form spores, although with low efficiency and viability 88 . However, without Dmc1, S. cerevisiae carry out almost no recombina- tion in meiosis and form extremely small numbers of spores 79 , leading to the proposal that Dmc1 is primarily responsible for interhomologue strand invasion during meiosis in S. cerevisiae. In A. thaliana, a mutation in RAD51 has been isolated that does not affect any mitotic functions, but has defective chromosome pairing and synapsis dur- ing meiosis 89 . A mutation in DMC1 results in random chromosome segregation and a residual fertility of 1.5% of the wild type 90 . In mice, homozygous Rad51 loss-of-function mutation results in early embryonic lethality 91,92 , whereas Dmc1 ?/? mice are infertile and their chromosomes do not synapse 93 . So, in many organisms, it seems that although RAD51 has a role in both mitotic and meiotic homologous recombina- tion, DMC1 is essential and specific for homologous recombination in meiosis. The Mnd1?Hop2 complex has recently been characterized as comprising meiotic genes encoding proteins that form a heterodimer, probably through predicted coiled-coil domains 94,95 . The proteins local- ize to meiotic chromosomes 95,96 . mnd1 and hop2 mutants in S. cerevisiae have a similar phenotype to dmc1 mutants, in that very little interhomologue strand invasion is detected 97 . Like Dmc1 ?/? mice, Hop2 ?/? mice are completely infertile, but do not have any overt mitotic defects 98 . Spermatogenesis in Hop2 ?/? mice reaches only the primary spermatocyte stage, which is consistent with a defect before the first meiotic nuclear division; this is consistent with what has been observed in S. cerevisiae. hop2-mutant S. cerevisiae show extensive synapsis of chromosomes, as measured by staining for the synaptonemal complex component Zip1 (molecular ZIPper), but this synapsis is non-homologous 96 . Although synapsis (presumably non-homologous) is extensive in an mnd1 mutant, 16 fully synapsed bivalents were never observed 99,100 . In contrast to the extensive synapsis in a hop2 mutant in S. cerevisiae, limited synapsis is seen in chromosome spreads of Hop2 ?/? mouse spermatocytes. Only 25% of cells had undergone more than 10% synapsis, most of which was non-homologous 98 . Therefore, mutations that affect Mnd1?Hop2 cause a synapsis defect in both yeast and mice. Interestingly, synapsis in a mouse Hop2 ?/? null mutant is more severely affected than it is in a Dmc1 ?/? mutant, in which 61% of cells have undergone at least 10% synapsis 98 . The Hop2 ?/? Dmc1 ?/? double mutant resembles the Hop2 ?/? mutant, with 26% of these cells having undergone 10% or more synapsis, which implies that HOP2 functions upstream of DMC1. However, in S. cerevisiae, hop2 dmc1 or hop2 rad51 double mutants have 40?50% pairing for a given chro- mosome, similar to the homologue pairing observed in a dmc1 or rad51 single mutant, and better than the 20% that is observed in a hop2 single mutant. This result indicates that in terms of homologue pairing, Hop2 might function downstream of Dmc1. Although these results might indicate true species differences in the order of action of Hop2 and Dmc1, another interpretation is that Mnd1?Hop2 might have roles both upstream and downstream of Dmc1. This issue will need to be resolved in future experiments. A com- mon feature of the mnd1?hop2 mutant phenotype in yeast and mice is a defect in homologue pairing and synaptonemal complex formation between the wrong chromosomes. NATURE REVIEWS | GENETICS VOLUME 6 | JUNE 2005 | 483 REVIEWS nrg0605-hawle.indd 483nrg0605-hawle.indd 483 17/5/05 5:20:59 pm17/5/05 5:20:59 pm � 2005 Nature Publishing Group Rad51-mediated homolgy search Dmc1?Mei5? Sae3-mediated stable strand invasion Mnd1?Hop2Dmc1?Mei5?Sae3 Rad51 Mode 1 (e.g. flies, worms) Mode 2 (e.g. yeast, mice, humans) DSB-independent homologue matching Low ectopic recombination No Mnd1?Hop2 Recombination dependent on Rad51 DSB-dependent homologue matching High ectopic recombination (yeast) Mnd1?Hop2 Recombination dependent on Dmc1 It has been proposed that Mnd1?Hop2 might cooperate with Dmc1 to promote stable strand inva- sion 97 . Dmc1 and Rad51 form foci in S. cerevisiae mnd1 and hop2 mutants 95,97,100 . Foci of recombination proteins (RAD51, DMC1 and RPA) were also observed on chromosomes of the mouse Hop2 ?/? mutant 98 . So recombination proteins can still assemble onto DNA, but they seem to be unable to mediate a successful extensive homology search or to catalyse stable strand invasion. Overexpression of Rad51 (but not Dmc1) sup- presses defects in meiotic recombination in S. cerevisiae hop2 mutants 101 . Taken together, these results indicate that there might be two pathways for recombination- mediated pairing of homologues ? one that depends on Rad51 to sense homology and one in which Dmc1 depends on Mnd1?Hop2 to ensure correct pairing and recombination 101 (FIG. 5). Consistent with this proposal, a recent study showed that a purified Mnd1?Hop2 heterodimer stimulates the in vitro strand-invasion activity of Dmc1 by threefold or more 94,120 . However, in cytological studies, Mnd1?Hop2 does not significantly co-localize with Rad51 (which does co- localize with Dmc1), arguing against a direct interac- tion of Mnd1?Hop2 with recombination proteins in vivo 100 . The issue of direct versus indirect interactions between recombinases and Mnd1?Hop2 will have to be determined in future experiments. Not all organisms have DMC1, HOP2 and MND1 orthologues. Although mammals, plants, protists and yeasts possess these genes, worms, flies and Neurospora crassa do not. This phyletic pattern correlates with the requirement for DSBs for synapsis and synaptonemal complex formation. Synaptonemal complex forma- tion seems to be dependent on DSBs in yeast and mice 5,10,12,102 . By contrast, in C. elegans and D. mela- nogaster, synaptonemal complexes form efficiently in the absence of the respective Spo11 homologues of these species uniF6AEREFS 14,15uniF6AF. As discussed earlier, fly and worm chromosomes have specific DNA sequences that can facilitate pairing in cis 24,103,104 . Furthermore, somatic pairing of fly chromosomes might alleviate the need for other pairing mechanisms during meiosis. Although it is possible that flies and worms have genes that serve a similar function to Dmc1?Mnd1?Hop2 that cannot be identified on the basis of sequence similarity, it seems more likely that pairing centres and/or somatic pairing can provide effective substitutes for Dmc1?Mnd1? Hop2-mediated pairing and recombination. In these organisms, meiotic recombination depends on Rad51 homologues. Mei5?Sae3 assists Dmc1. Mei5 and Sae3 form a heterodimer during meiosis that has been proposed to interact with Dmc1 uniF6AEREFS 105,106uniF6AF. In mei5, sae3 or dmc1 mutants, Rad51 associates with chromosomes, but does not seem to subsequently disassociate from them. Sporulation, spore viability and crossing over are reduced to similar levels in all three mutants. All three proteins co-localize on meiotic chromosomes and their localization is mutually dependent. Taken together, these results indicate that Mei5?Sae3 complexes are Dmc1-specific accessory factors required for catalytic and structural roles in interhomologue recombination during meiosis. Genome integrity in the face of high levels of recombi- nation. In S. cerevisiae, recombination rates are elevated about 1,000-fold in meiosis as compared with mitosis 107 . Surprisingly, recombination between homologous sequences located on homologous chromosomes is not particularly favoured over recombination between homologous sequences on non-homologous chromo- somes 107?109 , making recombination between dispersed homologous elements a rather frequent event in S. cerevisiae. These types of event could potentially be lethal if a translocation, or a dicentric or acentric chro- mosome is created. However, ectopic recombination during meiosis in flies is infrequent 110?113 . The rapid and DSB-independent synapsis and formation of synapton- emal complexes in flies and worms might help prevent the recombination of homologous sequences on non-homologous chromosomes in these organisms. Figure 5 | A model for Rad51, Dmc1?Mei5?Sae3 and Mnd1?Hop2 function in meiosis. Dmc1?Mei5?Sae3 (a recombination protein complex) assembles on 3? ssDNA on one side of a double-strand break (DSB) and the recombination protein Rad51 assembles on the other side. Dmc1?Mei5?Sae3 and Rad51 might be present in multiple copies, in multiprotein complexes or in different multimeric forms. In this model, the Rad51-bound end is primarily involved in the homology search that identifies the homologous chromosome. The interaction between homologous chromosomes is stabilized through interactions between Mnd1?Hop2 complexes that are likely to be present in multiple copies. Once the homologues are engaged in a stable interaction, Dmc1?Mei5?Sae3 can initiate a stable strand invasion. At this point, the Rad51-containing complex might dissociate from the DNA. Figure 6 | A comparison of the main features of recombination-dependent (mode 2) and independent (mode 1) matching and locking of homologous chromosomes. 484 | JUNE 2005 | VOLUME 6 www.nature.com/reviews/genetics REVIEWS nrg0605-hawle.indd 484nrg0605-hawle.indd 484 17/5/05 5:21:02 pm17/5/05 5:21:02 pm � 2005 Nature Publishing Group FLUORESCENCE IN SITU HYBRIDIZATION (FISH). A technique in which a fluorescently labelled DNA probe is used to hybridize with and therefore detect a particular chromosome or gene with the help of fluorescence microscopy. The synapsis of non-homologous chromosomes in a hop2 mutant in S. cerevisiae indicates that Mnd1?Hop2 might normally promote interactions that prevent recombination between non-homologous chromo- somes. It has recently been shown that although ectopic recombination between homologous sequences can be detected in dmc1, mnd1 and rad51 single mutants, mnd1 rad51 and hop2 rad51 double mutants have much higher levels of ectopic recombination between repetitive sequences, indicating that the proteins that these genes encode might normally have a synergistic role in preventing these types of potentially deleterious events (Henry, Rice and J.L.G., unpublished observa- tions). Therefore, Mnd1?Hop2, in conjunction with recombination proteins, might provide an alternative mechanism for helping to prevent ectopic events in the absence of rapid and DSB-independent synapsis. Challenges for the future Although all sexually reproducing organisms must match, lock and segregate their homologues during meiosis, it is clear that some organisms rely on certain mechanisms more than others. One of the biggest dif- ferences is that some organisms rely on recombination for synapsis (yeast and mice) whereas others apparently have other mechanisms that allow for synapsis in the absence of recombination (worms and flies, see FIG. 6). Still others, such as S. pombe and male D. melanogaster, do not form synaptonemal complexes at all. Why and how these differences have evolved is an intriguing question, and one for which there is no clear expla- nation. Even within a species there can be extreme differences in meiosis between the sexes, such as the difference between male D. melanogaster, which does not have recombination or synaptonemal complexes on any chromosome, and female D. melanogaster, which has recombination on at least two and usually three of the four chromosomes. However these dif- ferences arose, the choice of model system helps to focus studies on specific mechanisms 114 . As meiosis is studied in more model systems, we will come to have a better understanding and appreciation of each mechanism. In terms of physical assays for recombination intermediates that are currently available for in vivo studies uniF6AETABLE 1uniF6AF, it is possible to detect DSBs, at least in yeast. The next detectable intermediate is stable strand invasion. Currently, there is no simple physical assay to monitor the progress of the homology search. It is pos- sible to look at pairing of specific loci by FLUORESCENCE IN SITU HYBRIDIZATION (FISH) in chromosome spreads 115 uniF6AETABLE 1uniF6AF. Unfortunately, this method can give differ- ent results depending on the spreading technique used, the specific loci monitored, and the time at which the assay is carried out. It is also possible to look at pairing by Cre-mediated recombination of loxP sites placed throughout the genome 116,117 . This method can also yield different results depending on the sites that are being monitored. 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Online links DATABASES The following terms in this article are linked online to: Entrez: http://www.ncbi.nlm.nih.gov/entrez HIM-17 | rec12 | Spo11 OMIM: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=OMIM Down syndrome | Klinefelter syndrome | Turner syndrome FURTHER INFORMATION Stowers Institute Research Team web site: http://www.stowers-institute.org/labs/ResearchTeam.asp Access to this interactive links box is free online. NATURE REVIEWS | GENETICS VOLUME 6 | JUNE 2005 | 487 REVIEWS nrg0605-hawle.indd 487nrg0605-hawle.indd 487 17/5/05 5:21:09 pm17/5/05 5:21:09 pm "
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