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A role for recombination in centromere function
Opinion
A role for recombination in centromere function Ramsay J. McFarlane1 and Timothy C. Humphrey2 1
North West Cancer Research Fund Institute, Bangor University, Memorial Building, Deiniol Road, Bangor, Gwynedd, LL57 2UW, UK 2 Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ, UK
Centromeres are essential for chromosome segregation during both mitosis and meiosis. There are no obvious or conserved DNA sequence motif determinants for centromere function, but the complex centromeres found in the majority of eukaryotes studied to date consist of repetitive DNA sequences. A striking feature of these repeats is that they maintain a high level of inter-repeat sequence identity within the centromere. This observation is suggestive of a recombination mechanism that operates at centromeres. Here we postulate that interrepeat homologous recombination plays an intrinsic role in centromere function by forming covalently closed DNA loops. Moreover, the model provides an explanation of why both inverted and direct repeats are maintained and how they contribute to centromere function. Repetitive DNA at centromeres: a reason Centromeres are the regions of eukaryotic chromosomes that provide the docking sites for spindle microtubules during mitosis and meiosis (Figure 1a). Centromeres tend to be relatively large in size, with the point centromeres of Saccharomyces cerevisiae being notable exceptions. Whereas DNA sequence per se does not appear to govern centromere function, complex centromeres have one feature in common – they consist of repetitive DNA sequences, and these can be found in both inverted and direct configurations. Furthermore, centromeres have a unique chromatin structure and associate with specific proteins including the histone H3 variant CENP-A (centromere protein A). The lack of DNA sequence specificity at centromeres has led to the widely accepted dogma that the key feature for establishment of centromere function is the ability of a region of DNA to provide a platform for the creation of the unique CENP-A-containing chromatin [1– 7]. However, current proposals do not address the presence of repetitive DNA sequences within centromeres; indeed, studies of neocentromeres have shifted interest away from the possible importance of repetitive DNA. Here we present evidence for a new model in which we propose that inter-repeat homologous recombination plays a role in forming structures required for efficient centromere function. This model accounts for the need for repeat DNA sequences for full centromere function. Corresponding author: McFarlane, R.J. ([email protected]).
Fission yeast centromeres: a paradigm for centromeric inter-repeat recombination Studies of the fission yeast Schizosaccharomyces pombe have provided evidence for models explaining how complex centromeres function [1,8]. The three S. pombe centromeres range in size from approximately 30 kb to 110 kb and are made of inverted repeat sequences with a unique central core region (Figure 1b) [9]. A remarkable feature of the DNA sequences in the S. pombe centromeric repeats (and those of other complex centromeres), and that has largely eluded comment or explanation, is that the inverted repeat sequences within an individual centromere exhibit little or no divergence (Figure 1b; although they diverge between centromeres) [10]. Such extensive identity is only plausibly explained by there being genetic ‘crosstalk’ between these sequences in the form of gene-conversion recombination. This would imply that recombination occurs between inverted repeat structures on a frequent enough basis to eliminate any divergence generated by random mutations within either repeat. Sequence analysis of the inner most repeat (imr) of cen1 (locus designation for centromere 1) from distinct S. pombe strains demonstrated that imr sequences exhibit divergence between strains, but not between imrs within the same centromere [10]. Indeed, this study further demonstrated that the inter-strain divergence at the imr regions was similar to non-centromeric regions, indicating that sequence conservation per se is not crucial. Such an intra-genomic sequence maintenance mechanism is not without precedent. Fission yeast tRNA genes have been proposed to maintain sequence identity by a mechanism involving gene conversion between distantly located tRNA gene repeats [11–13]. Interestingly, tRNA genes are located within the identical inverted-repeat sequences of the fission yeast centromeres (Figure 1b). Therefore, it is possible that the mechanisms driving inter-tRNA gene conversion could provide the basis for inter-repeat recombination at centromeres. Indeed, tRNA genes can serve as barriers to the progression of DNA replication forks [14]. Such barriers possess recombination initiator potential and are associated with genome fragile sites and evolutionary break points in yeast [15–19]. Under normal conditions the recombinogenic potential of tRNA genes appears to be low. However, when proteins associated with the replisome progression complex are mutated, the recombinogenic potential of tRNA genes becomes significant [19]. Fission yeast
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Figure 1. Inverted repeats of fission yeast centromeres recombine to form a covalent ring structure. This figure is a schematic representation of the model for how inverted repeats within centromeres can associated by inter-repeat recombination to form a covalently closed stem structure that generates a loop of the DNA region between the inverted repeats. This model is illustrated by the fission yeast centromere 1 that consists of an inverted-repeat configuration. (a) Centromeres of sister chromatids (yellow circles) provide the docking sites for spindle microtubules (black lines). Arrows show the direction of the force exerted by the microtubules. (b) Schematic representation of the 30 kb centromere I (cen1) of S. pombe. The central core (yellow) is flanked by inner most repeats imrL (left) and imrR (right) in green, and outer most repeats (otrs) dg (blue) and dh (pink). The vertical lines within the imrs and at the outer left flank represent tRNA genes. (c) A schematic representation of a proposed covalent ring structure for S. pombe cen1. The black line represents the duplex DNA from a single chromatid and the X represents a covalent recombination intermediate (single or double Holliday junction). The relative positions of the repeat sequences are shown as in (b). Nucleosomes are represented as spheres. Nucleosomes containing histone H3 are represented by blue spheres and nucleosomes containing Cnp1 (CENP-A) are represented by yellow spheres.
centromeres are replicated early during S-phase [20] and it is possible that replication complexes, that are established early in S-phase, are distinctly modulated to stimulate the formation of recombination-initiating lesions at centromeric tRNA genes. It is therefore possible that tRNA gene-initiated interrepeat recombination accounts for the unexplained observation that inverted-repeat identity in fission yeast centromeres is maintained. Indeed, the tRNA genes within the imr regions of fission yeast cen1 are required for full centromere function [21]. One could therefore hypothesise that the establishment of inter-repeat recombination intermediates plays a role in centromere function. As such, recombination intermediates such as Holliday junctions could provide covalent linkage in a stem structure that then results in a covalently closed loop (CCL) within the centromere, with Cnp1 (CENP-A) located at the loop (Figure 1c). Such a loop structure has parallels with a cohesinmediated stem-loop structure that has been proposed for the point centromeres and the pericentric regions of S. cerevisiae, and the more complex centromeres of S. pombe, in which the stem is generated by intra-chromatid cohesion, rather than by a covalently linked recombination reaction [22–24]. Such a cohesion-mediated structure might provide a paradigm for an evolutionary precursor to larger recombination-associated centromeres. This proposal opens up the possibility that sister centromere CCLs could become catenated, resulting in covalently linked sister chromatids that could then be resolved by the activity of topoisomerases; indeed, it has been proposed that topoisomerases provide a role in resolving inter-sister associations during the metaphase to ana210
phase progression [25]. Interestingly, a catenated loop configuration has parallels with catenated circular bacterial chromosomes that are resolved via site-specific recombination [26,27]. Recent work has demonstrated that functional centromeres can be established in a fission yeast plasmid containing the central core region of centromere 2 (cen) provided that heterochromatin flanks one side of the core [28]. This indicates that the outer most repeat (otr) regions (Figure 1) per se are not required for centromere function during mitosis and that these regions serve to provide a platform for the essential heterochromatin component of centromeres [28]. However, the cen2 central core region employed in these studies retains the cen2 core-associated inverted repeats (CARs) [28,29] thereby providing sufficient homology for recombination to form CCLs. Interestingly, these plasmids only have heterochromatin induced on one side of the central core element, the region associated with Cnp1 (CENP-A). Despite this asymmetric heterochromatin, Cnp1 distribution was limited to the central core region, indicating that another factor determines Cnp1 distribution. It is possible that structural intermediates formed by recombination events between the core-flanking inverted repeats might provide the boundaries to Cnp1 distribution. Indeed, in 2D gel analysis of genomic cen2, the region containing the central core and the CARs displayed unique structures that did not correspond to known DNA replication intermediates [30], consistent with complex recombination intermediates being present. In support of the formation of recombination intermediates at centromeres, recent work from mammals has identified HJURP (Holliday junction recognising protein), a novel Holliday junction binding protein associated with
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centromere structure, and that serves as a CENP-A chaperone [31–34]. The specificity of this protein for Holliday junctions remains unclear, but this model offers an explanation for the targeting of the CENP-A chaperone to centromeres. Evidence from isochromosome formation? Isochromosomes are generated when one chromosome arm is replaced by an inverted duplication of the other arm. Two recent reports showed that isochromosome formation is associated with centromeric recombination in fission yeast [35,36]. Analysing gross chromosomal rearrangements within a non-essential mini chromosome, Nakagawa and co-workers [35] found evidence for spontaneous isochromosome formation. In a separate study [36], induction of a DNA double-strand break (DSB) distal to the centromere within one arm of a non-essential mini chromosome was able to initiate isochromosome formation. Remarkably, in both studies the isochromosome breakpoints mapped to very similar regions in the mini chromosome centromeres. To account for the observation that a DSB located a considerable distance from the centromere could induce an isochromosome breakpoint within the centromere, a ‘break–chew and copy’ model was proposed in which extensive DSB end processing results in the loss of the broken arm. It was proposed that the degradation of the break (the ‘chew’) extended to the centromeric regions, where the inverted repeats in the imr (Figure 1) provide the substrate for a break-induced replication event within the centromere. Curiously, both studies found elevated isochromosome formation in mutants defective for Rad51, the RecA-like protein that mediates the essential strand invasion step of homologous recombination [37]. The absence of Rad51 is predicted to result in elevated levels of failed recombinationmediated DNA repair of broken chromosomes, thus leading to a greater number of broken ends undergoing extensive end processing [36]. In this respect, resection through to a centromeric structure in the form of a physical recombination intermediate between the centromeric imr repeats might facilitate Rad51-independent break-induced replication, and subsequently isochromosome formation. In addition, Nakagawa and co-workers found high levels of minichromosome loss and sensitivity to the microtubuledestabilising drug thiabendazole in Rad51-deficient cells, phenotypes associated with centromeric dysfunction, indicating a link to recombination and centromere function [35]. Multiple CCLs at large centromeres How might this model be applied to the large centromeres of metazoans such as humans? In such centromeres coalignment of direct repeat sequences cannot form the stem– loop structures that can be formed by inverted repeats (Figure 1c). However, recombination is not restricted to inverted repeats; recombination between direct repeats would still generate CCLs by co-alignment of two of the direct repeats, and thereby generate a loop of the DNA between them (Figure 2). A non-covalently closed looping model for complex direct-repeat centromeres has been developed to explain the fact that CENP-A is not laid down continuously on centromeres but that stretches of CENP-A are instead broken up with stretches of
Figure 2. Inter-direct repeat recombination could generate covalently closed rings within large complex centromeres. This is a schematic of how large centromeres consisting of multiple direct repeats, as found in human centromeres, might generate loop structures via recombination between homologous direct repeats. Moreover, it demonstrates how the pattern of distribution of the histone H3 variant CENP-A fits with a recombination-mediated loop model. Top: direct repeats of large centromeres are schematically represented by the blue arrows over the DNA (black line). The blue and yellow spheres below the line represent the discontinuous distribution of nucleosomes containing either histone H3 (blue) or the histone H3 variant CENP-A (yellow). Bottom: homologous recombination between direct repeats drives the formation of covalently closed rings along the centromeric DNA. The X represents a covalent recombination intermediate (single or double Holliday junction).
H3-containing nucleosomes (Figure 2) [3,38]. Such a looping model is consistent with loops being converted to a series of CCLs by inter-repeat recombination. Metazoans might require a threshold number of these loop structures to ensure that a fully functional centromere is developed. Crossover recombination between these direct repeats would result in the release of the ring from the body of the chromosome to form extrachromosomal circular molecules. This could result in the shortening of centromeres with progressive numbers of cell divisions, thereby resulting in shorter centromeres in older cells. Consistent with this hypothesis, ‘centromere deterioration’ has been observed in aged female humans [39]. Moreover, extrachromosomal circles containing centromeric DNA sequences have been identified in human cells [40–43]. It is possible that shorter centromeres could be functionally compromised, thus leading to increased aneuploidy as observed in older cells. Alternatively, these ‘old’ cells with shorter centromeres might be compromised in kinetochore attachment, thus engaging the mitotic spindle checkpoint, and subsequently failing to retain proliferative potential. Consistent with this idea, a ‘kinetochore maintenance mechanism’ has been postulated [44]. This mechanism might ensure there is a bias to non-crossover recombination. Dysfunction in such a maintenance mechanism could be linked to cross-over induced ‘centromere deterioration’. In addition, inter-sister chromatid recombination is higher 211
Opinion at centromeres than other heterochromatic regions such as telomeres, and loss of DNA methyl transferase activity, that is required for formation of heterochromatin at mammalian centromeres, results in centromeres becoming shorter [45]. These observations indicate that there is elevated recombination activity at mammalian centromeres, and these require heterochromatin to prevent recombination-dependent reduction of the number of centromeric repeats [45]. By contrast, single yeast cells with inverted-repeat centromeres (such as S. pombe), in which the number of cell divisions is potentially unlimited, have inverted centromeric repeats that will not be excised if recombination intermediates are resolved without crossing over. Neocentromeres Neocentromers are chromosomal regions that have the ability to provide the platform to form functional kinetochores when normal centromeres are lost or become dysfunctional. They have been induced in yeast species that have complex centromeres, and occur spontaneously in metazoans, including humans, where they are associated with a broad range of genetic diseases, including foetal abnormalities, mental retardation, skin pigment and skeletal abnormalities and cancers [46]. Whereas human neocentromeres are initially devoid of alphoid repeats (specific 171 bp repeats located within human centromeres), they appear to generate new areas of extensive repetitive DNA sequences over time, indicating that there is an advantage to acquiring an extensive repeat configuration for long-term fixing of neocentromeres [46]. Little is known about the fine structure of most neocentromeres. However, human neocentromeres that have been studied to a relatively high resolution are enriched for repetitive L1 long interspersed nuclear elements (LINEs) [47–49], indicating that they form at regions containing pre-existing repeats. DNA sequencing of a 80 kb neocentromere derived from human chromosome 10 revealed that although there were no classical alphoid repeats, there were a number of other repeat regions present [50]. These repeat-enriched regions might thus provide sufficient substrates for inter-repeat recombination to generate CCLs. Over time, these regions become fixed by acquiring further repeat elements by unknown mechanisms. It is interesting to note that primary human neocentromeres appear to bind less CENP-A relative to other human centromeres, a finding consistent with more limited repeats forming lower numbers of CENP-A containing loops [51]. In addition, in the majority of invertedduplication chromosomes the neocentromeres map close to the recombination site that generated the inversion, indicating they are associated with an inverted-repeat region [46]. This might indicate that, for neocentromere formation in humans, a recombination event between inverted repeats, creating a single CCL, is sufficient to establish neocentromere function, although long-term fixing might require the generation of more extensive repeats. Neocentromeres have also been generated in both S. pombe and Candida albicans [52,53]. In S. pombe deletion of cen1 results in the formation of rare survivors (<1 in 1,000) that have acquired neocentromeres adjacent to one 212
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of the telomeres [52]. Whether centromere function utilises telomeric sequence repeats or whether neocentromeres are associated with new repeat sequences in these telomereproximal regions remains unknown. As in S. cerevisiae, C. albicans kinetochores attach to only a single microtubule [54], and provide a possible paradigm for the evolutionary transition from simple point centromeres to more complex regional centromeres. C. albicans lacks centromeric heterochromatin and the eight chromosomes have a range of centromere types including point centromeres, inverted-repeat centromeres (that are relatively small compared to those of S. pombe) and simple direct-repeat centromeres [55]. The deletion of CEN5, an inverted-repeat centromere, results in the formation of neocentromeres at a number of distinct sites [53]. These neocentromeres share the common feature of being associated with a DNA repeat, and this might provide interchromatid repeat recombination substrates, consistent with the proposed model. Concluding remarks In summary, we present a model for the structure of complex centromeres that proposes a functional role for homologous recombination within centromeric DNA. There is a clear precedent for such a role for recombination in determining a key chromosomal structure, because it has been proposed for some time that recombination plays a role in the formation of T-loop structures at telomeres [56–58]. Our proposed model opens up new possibilities of how we view the function of centromeres beyond the simple regulation of chromatid separation at anaphase into control of the cell division potential of metazoan cells. Although we propose the existence of CCLs at complex centromeres, we make no explicit suggestion as to how these covalent rings might function, leaving open new questions for exploration. Moreover, it might simply be the case that such structures are not absolutely required, but instead serve to enhance centromere function. This model can be tested in a number of ways, including a more thorough analysis of unusual DNA structures found at some centromeres [29] and a more comprehensive analysis of the biochemical properties of centromere-associated proteins, such as HJURP – the Holliday junction binding protein and CENP-A chaperone. This model potentiates many new avenues for investigation and it is clear that establishing a role for recombination intermediates in centromere function will provide important insight into genome stability. Acknowledgements We would like to thank Edgar Hartsuiker, David Pryce, Jane Wakeman and Simon Whitehall for reviewing the manuscript.
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A role for recombination in centromere function Ramsay J. McFarlane1 and Timothy C. Humphrey2 1
North West Cancer Research Fund Institute, Bangor University, Memorial Building, Deiniol Road, Bangor, Gwynedd, LL57 2UW, UK 2 Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ, UK
Centromeres are essential for chromosome segregation during both mitosis and meiosis. There are no obvious or conserved DNA sequence motif determinants for centromere function, but the complex centromeres found in the majority of eukaryotes studied to date consist of repetitive DNA sequences. A striking feature of these repeats is that they maintain a high level of inter-repeat sequence identity within the centromere. This observation is suggestive of a recombination mechanism that operates at centromeres. Here we postulate that interrepeat homologous recombination plays an intrinsic role in centromere function by forming covalently closed DNA loops. Moreover, the model provides an explanation of why both inverted and direct repeats are maintained and how they contribute to centromere function. Repetitive DNA at centromeres: a reason Centromeres are the regions of eukaryotic chromosomes that provide the docking sites for spindle microtubules during mitosis and meiosis (Figure 1a). Centromeres tend to be relatively large in size, with the point centromeres of Saccharomyces cerevisiae being notable exceptions. Whereas DNA sequence per se does not appear to govern centromere function, complex centromeres have one feature in common – they consist of repetitive DNA sequences, and these can be found in both inverted and direct configurations. Furthermore, centromeres have a unique chromatin structure and associate with specific proteins including the histone H3 variant CENP-A (centromere protein A). The lack of DNA sequence specificity at centromeres has led to the widely accepted dogma that the key feature for establishment of centromere function is the ability of a region of DNA to provide a platform for the creation of the unique CENP-A-containing chromatin [1– 7]. However, current proposals do not address the presence of repetitive DNA sequences within centromeres; indeed, studies of neocentromeres have shifted interest away from the possible importance of repetitive DNA. Here we present evidence for a new model in which we propose that inter-repeat homologous recombination plays a role in forming structures required for efficient centromere function. This model accounts for the need for repeat DNA sequences for full centromere function. Corresponding author: McFarlane, R.J. ([email protected]).
Fission yeast centromeres: a paradigm for centromeric inter-repeat recombination Studies of the fission yeast Schizosaccharomyces pombe have provided evidence for models explaining how complex centromeres function [1,8]. The three S. pombe centromeres range in size from approximately 30 kb to 110 kb and are made of inverted repeat sequences with a unique central core region (Figure 1b) [9]. A remarkable feature of the DNA sequences in the S. pombe centromeric repeats (and those of other complex centromeres), and that has largely eluded comment or explanation, is that the inverted repeat sequences within an individual centromere exhibit little or no divergence (Figure 1b; although they diverge between centromeres) [10]. Such extensive identity is only plausibly explained by there being genetic ‘crosstalk’ between these sequences in the form of gene-conversion recombination. This would imply that recombination occurs between inverted repeat structures on a frequent enough basis to eliminate any divergence generated by random mutations within either repeat. Sequence analysis of the inner most repeat (imr) of cen1 (locus designation for centromere 1) from distinct S. pombe strains demonstrated that imr sequences exhibit divergence between strains, but not between imrs within the same centromere [10]. Indeed, this study further demonstrated that the inter-strain divergence at the imr regions was similar to non-centromeric regions, indicating that sequence conservation per se is not crucial. Such an intra-genomic sequence maintenance mechanism is not without precedent. Fission yeast tRNA genes have been proposed to maintain sequence identity by a mechanism involving gene conversion between distantly located tRNA gene repeats [11–13]. Interestingly, tRNA genes are located within the identical inverted-repeat sequences of the fission yeast centromeres (Figure 1b). Therefore, it is possible that the mechanisms driving inter-tRNA gene conversion could provide the basis for inter-repeat recombination at centromeres. Indeed, tRNA genes can serve as barriers to the progression of DNA replication forks [14]. Such barriers possess recombination initiator potential and are associated with genome fragile sites and evolutionary break points in yeast [15–19]. Under normal conditions the recombinogenic potential of tRNA genes appears to be low. However, when proteins associated with the replisome progression complex are mutated, the recombinogenic potential of tRNA genes becomes significant [19]. Fission yeast
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Figure 1. Inverted repeats of fission yeast centromeres recombine to form a covalent ring structure. This figure is a schematic representation of the model for how inverted repeats within centromeres can associated by inter-repeat recombination to form a covalently closed stem structure that generates a loop of the DNA region between the inverted repeats. This model is illustrated by the fission yeast centromere 1 that consists of an inverted-repeat configuration. (a) Centromeres of sister chromatids (yellow circles) provide the docking sites for spindle microtubules (black lines). Arrows show the direction of the force exerted by the microtubules. (b) Schematic representation of the 30 kb centromere I (cen1) of S. pombe. The central core (yellow) is flanked by inner most repeats imrL (left) and imrR (right) in green, and outer most repeats (otrs) dg (blue) and dh (pink). The vertical lines within the imrs and at the outer left flank represent tRNA genes. (c) A schematic representation of a proposed covalent ring structure for S. pombe cen1. The black line represents the duplex DNA from a single chromatid and the X represents a covalent recombination intermediate (single or double Holliday junction). The relative positions of the repeat sequences are shown as in (b). Nucleosomes are represented as spheres. Nucleosomes containing histone H3 are represented by blue spheres and nucleosomes containing Cnp1 (CENP-A) are represented by yellow spheres.
centromeres are replicated early during S-phase [20] and it is possible that replication complexes, that are established early in S-phase, are distinctly modulated to stimulate the formation of recombination-initiating lesions at centromeric tRNA genes. It is therefore possible that tRNA gene-initiated interrepeat recombination accounts for the unexplained observation that inverted-repeat identity in fission yeast centromeres is maintained. Indeed, the tRNA genes within the imr regions of fission yeast cen1 are required for full centromere function [21]. One could therefore hypothesise that the establishment of inter-repeat recombination intermediates plays a role in centromere function. As such, recombination intermediates such as Holliday junctions could provide covalent linkage in a stem structure that then results in a covalently closed loop (CCL) within the centromere, with Cnp1 (CENP-A) located at the loop (Figure 1c). Such a loop structure has parallels with a cohesinmediated stem-loop structure that has been proposed for the point centromeres and the pericentric regions of S. cerevisiae, and the more complex centromeres of S. pombe, in which the stem is generated by intra-chromatid cohesion, rather than by a covalently linked recombination reaction [22–24]. Such a cohesion-mediated structure might provide a paradigm for an evolutionary precursor to larger recombination-associated centromeres. This proposal opens up the possibility that sister centromere CCLs could become catenated, resulting in covalently linked sister chromatids that could then be resolved by the activity of topoisomerases; indeed, it has been proposed that topoisomerases provide a role in resolving inter-sister associations during the metaphase to ana210
phase progression [25]. Interestingly, a catenated loop configuration has parallels with catenated circular bacterial chromosomes that are resolved via site-specific recombination [26,27]. Recent work has demonstrated that functional centromeres can be established in a fission yeast plasmid containing the central core region of centromere 2 (cen) provided that heterochromatin flanks one side of the core [28]. This indicates that the outer most repeat (otr) regions (Figure 1) per se are not required for centromere function during mitosis and that these regions serve to provide a platform for the essential heterochromatin component of centromeres [28]. However, the cen2 central core region employed in these studies retains the cen2 core-associated inverted repeats (CARs) [28,29] thereby providing sufficient homology for recombination to form CCLs. Interestingly, these plasmids only have heterochromatin induced on one side of the central core element, the region associated with Cnp1 (CENP-A). Despite this asymmetric heterochromatin, Cnp1 distribution was limited to the central core region, indicating that another factor determines Cnp1 distribution. It is possible that structural intermediates formed by recombination events between the core-flanking inverted repeats might provide the boundaries to Cnp1 distribution. Indeed, in 2D gel analysis of genomic cen2, the region containing the central core and the CARs displayed unique structures that did not correspond to known DNA replication intermediates [30], consistent with complex recombination intermediates being present. In support of the formation of recombination intermediates at centromeres, recent work from mammals has identified HJURP (Holliday junction recognising protein), a novel Holliday junction binding protein associated with
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centromere structure, and that serves as a CENP-A chaperone [31–34]. The specificity of this protein for Holliday junctions remains unclear, but this model offers an explanation for the targeting of the CENP-A chaperone to centromeres. Evidence from isochromosome formation? Isochromosomes are generated when one chromosome arm is replaced by an inverted duplication of the other arm. Two recent reports showed that isochromosome formation is associated with centromeric recombination in fission yeast [35,36]. Analysing gross chromosomal rearrangements within a non-essential mini chromosome, Nakagawa and co-workers [35] found evidence for spontaneous isochromosome formation. In a separate study [36], induction of a DNA double-strand break (DSB) distal to the centromere within one arm of a non-essential mini chromosome was able to initiate isochromosome formation. Remarkably, in both studies the isochromosome breakpoints mapped to very similar regions in the mini chromosome centromeres. To account for the observation that a DSB located a considerable distance from the centromere could induce an isochromosome breakpoint within the centromere, a ‘break–chew and copy’ model was proposed in which extensive DSB end processing results in the loss of the broken arm. It was proposed that the degradation of the break (the ‘chew’) extended to the centromeric regions, where the inverted repeats in the imr (Figure 1) provide the substrate for a break-induced replication event within the centromere. Curiously, both studies found elevated isochromosome formation in mutants defective for Rad51, the RecA-like protein that mediates the essential strand invasion step of homologous recombination [37]. The absence of Rad51 is predicted to result in elevated levels of failed recombinationmediated DNA repair of broken chromosomes, thus leading to a greater number of broken ends undergoing extensive end processing [36]. In this respect, resection through to a centromeric structure in the form of a physical recombination intermediate between the centromeric imr repeats might facilitate Rad51-independent break-induced replication, and subsequently isochromosome formation. In addition, Nakagawa and co-workers found high levels of minichromosome loss and sensitivity to the microtubuledestabilising drug thiabendazole in Rad51-deficient cells, phenotypes associated with centromeric dysfunction, indicating a link to recombination and centromere function [35]. Multiple CCLs at large centromeres How might this model be applied to the large centromeres of metazoans such as humans? In such centromeres coalignment of direct repeat sequences cannot form the stem– loop structures that can be formed by inverted repeats (Figure 1c). However, recombination is not restricted to inverted repeats; recombination between direct repeats would still generate CCLs by co-alignment of two of the direct repeats, and thereby generate a loop of the DNA between them (Figure 2). A non-covalently closed looping model for complex direct-repeat centromeres has been developed to explain the fact that CENP-A is not laid down continuously on centromeres but that stretches of CENP-A are instead broken up with stretches of
Figure 2. Inter-direct repeat recombination could generate covalently closed rings within large complex centromeres. This is a schematic of how large centromeres consisting of multiple direct repeats, as found in human centromeres, might generate loop structures via recombination between homologous direct repeats. Moreover, it demonstrates how the pattern of distribution of the histone H3 variant CENP-A fits with a recombination-mediated loop model. Top: direct repeats of large centromeres are schematically represented by the blue arrows over the DNA (black line). The blue and yellow spheres below the line represent the discontinuous distribution of nucleosomes containing either histone H3 (blue) or the histone H3 variant CENP-A (yellow). Bottom: homologous recombination between direct repeats drives the formation of covalently closed rings along the centromeric DNA. The X represents a covalent recombination intermediate (single or double Holliday junction).
H3-containing nucleosomes (Figure 2) [3,38]. Such a looping model is consistent with loops being converted to a series of CCLs by inter-repeat recombination. Metazoans might require a threshold number of these loop structures to ensure that a fully functional centromere is developed. Crossover recombination between these direct repeats would result in the release of the ring from the body of the chromosome to form extrachromosomal circular molecules. This could result in the shortening of centromeres with progressive numbers of cell divisions, thereby resulting in shorter centromeres in older cells. Consistent with this hypothesis, ‘centromere deterioration’ has been observed in aged female humans [39]. Moreover, extrachromosomal circles containing centromeric DNA sequences have been identified in human cells [40–43]. It is possible that shorter centromeres could be functionally compromised, thus leading to increased aneuploidy as observed in older cells. Alternatively, these ‘old’ cells with shorter centromeres might be compromised in kinetochore attachment, thus engaging the mitotic spindle checkpoint, and subsequently failing to retain proliferative potential. Consistent with this idea, a ‘kinetochore maintenance mechanism’ has been postulated [44]. This mechanism might ensure there is a bias to non-crossover recombination. Dysfunction in such a maintenance mechanism could be linked to cross-over induced ‘centromere deterioration’. In addition, inter-sister chromatid recombination is higher 211
Opinion at centromeres than other heterochromatic regions such as telomeres, and loss of DNA methyl transferase activity, that is required for formation of heterochromatin at mammalian centromeres, results in centromeres becoming shorter [45]. These observations indicate that there is elevated recombination activity at mammalian centromeres, and these require heterochromatin to prevent recombination-dependent reduction of the number of centromeric repeats [45]. By contrast, single yeast cells with inverted-repeat centromeres (such as S. pombe), in which the number of cell divisions is potentially unlimited, have inverted centromeric repeats that will not be excised if recombination intermediates are resolved without crossing over. Neocentromeres Neocentromers are chromosomal regions that have the ability to provide the platform to form functional kinetochores when normal centromeres are lost or become dysfunctional. They have been induced in yeast species that have complex centromeres, and occur spontaneously in metazoans, including humans, where they are associated with a broad range of genetic diseases, including foetal abnormalities, mental retardation, skin pigment and skeletal abnormalities and cancers [46]. Whereas human neocentromeres are initially devoid of alphoid repeats (specific 171 bp repeats located within human centromeres), they appear to generate new areas of extensive repetitive DNA sequences over time, indicating that there is an advantage to acquiring an extensive repeat configuration for long-term fixing of neocentromeres [46]. Little is known about the fine structure of most neocentromeres. However, human neocentromeres that have been studied to a relatively high resolution are enriched for repetitive L1 long interspersed nuclear elements (LINEs) [47–49], indicating that they form at regions containing pre-existing repeats. DNA sequencing of a 80 kb neocentromere derived from human chromosome 10 revealed that although there were no classical alphoid repeats, there were a number of other repeat regions present [50]. These repeat-enriched regions might thus provide sufficient substrates for inter-repeat recombination to generate CCLs. Over time, these regions become fixed by acquiring further repeat elements by unknown mechanisms. It is interesting to note that primary human neocentromeres appear to bind less CENP-A relative to other human centromeres, a finding consistent with more limited repeats forming lower numbers of CENP-A containing loops [51]. In addition, in the majority of invertedduplication chromosomes the neocentromeres map close to the recombination site that generated the inversion, indicating they are associated with an inverted-repeat region [46]. This might indicate that, for neocentromere formation in humans, a recombination event between inverted repeats, creating a single CCL, is sufficient to establish neocentromere function, although long-term fixing might require the generation of more extensive repeats. Neocentromeres have also been generated in both S. pombe and Candida albicans [52,53]. In S. pombe deletion of cen1 results in the formation of rare survivors (<1 in 1,000) that have acquired neocentromeres adjacent to one 212
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of the telomeres [52]. Whether centromere function utilises telomeric sequence repeats or whether neocentromeres are associated with new repeat sequences in these telomereproximal regions remains unknown. As in S. cerevisiae, C. albicans kinetochores attach to only a single microtubule [54], and provide a possible paradigm for the evolutionary transition from simple point centromeres to more complex regional centromeres. C. albicans lacks centromeric heterochromatin and the eight chromosomes have a range of centromere types including point centromeres, inverted-repeat centromeres (that are relatively small compared to those of S. pombe) and simple direct-repeat centromeres [55]. The deletion of CEN5, an inverted-repeat centromere, results in the formation of neocentromeres at a number of distinct sites [53]. These neocentromeres share the common feature of being associated with a DNA repeat, and this might provide interchromatid repeat recombination substrates, consistent with the proposed model. Concluding remarks In summary, we present a model for the structure of complex centromeres that proposes a functional role for homologous recombination within centromeric DNA. There is a clear precedent for such a role for recombination in determining a key chromosomal structure, because it has been proposed for some time that recombination plays a role in the formation of T-loop structures at telomeres [56–58]. Our proposed model opens up new possibilities of how we view the function of centromeres beyond the simple regulation of chromatid separation at anaphase into control of the cell division potential of metazoan cells. Although we propose the existence of CCLs at complex centromeres, we make no explicit suggestion as to how these covalent rings might function, leaving open new questions for exploration. Moreover, it might simply be the case that such structures are not absolutely required, but instead serve to enhance centromere function. This model can be tested in a number of ways, including a more thorough analysis of unusual DNA structures found at some centromeres [29] and a more comprehensive analysis of the biochemical properties of centromere-associated proteins, such as HJURP – the Holliday junction binding protein and CENP-A chaperone. This model potentiates many new avenues for investigation and it is clear that establishing a role for recombination intermediates in centromere function will provide important insight into genome stability. Acknowledgements We would like to thank Edgar Hartsuiker, David Pryce, Jane Wakeman and Simon Whitehall for reviewing the manuscript.
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