Repeat instability at human minisatellites arising from meiotic recombination.

Little is known about the role of meiotic recombination processes such as unequal crossover in driving instability at tandem repeat DNA. Methods have therefore been developed to detect meiotic crossovers within two different GC-rich minisatellite repeat arrays in humans, both in families and in sperm DNA. Both loci normally mutate in the germline by complex conversion-like transfer of repeats between alleles. Analysis shows that inter-allelic unequal crossovers also occur at both loci, although at low frequency, to yield simple recombinant repeat arrays with exchange of flanking markers. Equal crossovers between aligned alleles, resulting in recombinant alleles but without change in repeat copy number, also occur in sperm at a similar frequency to unequal crossovers. Both crossover and conversion show polarity in the repeat array and are co-suppressed in an allele showing unusual germline stability. This provides evidence that minisatellite conversion and crossover arise by a common mechanism, perhaps by alternative processing of a meiotic recombination initiation complex, and implies that minisatellite instability is a by-product of meiotic recombination in repeat DNA. While minisatellite recombination is infrequent, crossover rates indicate that the unstable end of a human minisatellite can act as a recombination warm-spot, even between sequence-heterologous alleles.     

Influences of array size and homogeneity on minisatellite mutation.

Unstable minisatellites display high frequencies of spontaneous gain and loss of repeats in the human germline. Most length changes arise through complex recombination events including intra-allelic duplications/deletions and inter-allelic transfers of repeats. Definition of the factors modulating instability requires both measurement of mutation rate and detailed analysis of mutant structures at the level of individual alleles. We have measured mutation rates in sperm for a wide range of alleles of the highly unstable human minisatellite CEB1. Instability varies by three orders of magnitude between alleles and increases steadily with the size of the tandem array. Structural analysis of mutant molecules derived from six alleles revealed that it is the rate of intra-allelic rearrangements which increases with array size and that intra-allelic duplication events tend to cluster within homogeneous segments of alleles; both phenomena resemble features of trinucleotide repeat instability. In contrast, inter-allelic transfers occur at a fairly constant rate, irrespective of array length, and show a mild polarity towards one end of the minisatellite, suggesting the possible influence of flanking DNA on these conversion-like events.     

Complex minisatellite rearrangements generated in the total or partial absence of Rad27/hFEN1 activity occur in a single generation and are Rad51 and Rad52 dependent

Genomes contain tandem repeat blocks that are at risk of expansion or contraction. The mechanisms of destabilization of the human minisatellite CEB1 (arrays of 36- to 43-bp repeats) were investigated in a previously developed model system, in which CEB1-0.6 (14 repeats) and CEB1-1.8 (42 repeats) alleles were inserted into the genome of Saccharomyces cerevisiae. As in human cells, CEB1 is stable in mitotically growing yeast cells but is frequently rearranged in the absence of the Rad27/hFEN1 protein involved in Okazaki fragments maturation. To gain insight into this mode of destabilization, the CEB1-1.8 and CEB1-0.6 human alleles and 47 rearrangements derived from a CEB1-1.8 progenitor in rad27Delta cells were sequenced. A high degree of polymorphism of CEB1 internal repeats was observed, attesting to a large variety of homology-driven rearrangements. Simple deletion, double deletion, and highly complex events were observed. Pedigree analysis showed that all rearrangements, even the most complex, occurred in a single generation and were inherited equally by mother and daughter cells. Finally, the rearrangement frequency was found to increase with array size, and partial complementation of the rad27Delta mutation by hFEN1 demonstrated that the production of novel CEB1 alleles is Rad52 and Rad51 dependent. Instability can be explained by an accumulation of unresolved flap structures during replication, leading to the formation of recombinogenic lesions and faulty repair, best understood by homology-dependent synthesis-strand displacement and annealing.     

Somatic versus germline mutation processes at minisatellite CEB1 (D2S90) in humans and transgenic mice

The most variable human minisatellites show extreme germline instability dominated by complex intra-allelic rearrangements plus a lower frequency of inter-allelic transfers of repeat units. In contrast, little is known about somatic instability at such loci. We have therefore used single-molecule PCR to analyze mutation at minisatellite CEB1 (D2S90) in human blood DNA. Somatic mutants were rare and involved only relatively simple intra-allelic events, with no bias toward expansions, in sharp contrast to the complex gain-biased rearrangements seen in sperm. Somatic and germline mutation processes were further analyzed in mice transgenic for a cosmid insert containing CEB1. Mutant molecules in transgenic sperm and blood were detected but only at the low frequencies seen in human blood and arose mainly by simple duplications and deletions as seen for somatic mutations in human. These data suggest distinct pathways for germline and somatic CEB1 mutations with germline instability involving recombination-based repair of meiotic double-strand breaks and somatic mutation arising by replication slippage or mitotic recombination. The problem of transferring germline-specific features of minisatellite instability from human to mouse suggests, with other recent observations, that long-range chromatin conformation may be required for the recombination-based mode of germline instability at human minisatellites.     

Repeat unit sequence variation in minisatellites: a novel source of DNA polymorphism for studying variation and mutation by single molecule analysis

Variation in internal minisatellite structure can be analyzed by mapping variant repeat units within amplified alleles. A system capable of distinguishing greater than 10(70) allelic states at the human hypervariable locus D1S8 has been developed. Population surveys of internal allelic structure indicate that D1S8 alleles evolve rapidly along haploid chromosome lineages. Internal mapping of deletion mutant alleles physically selected from genomic DNA provides further evidence that germline and somatic mutations altering the number of allelic repeat units seldom if ever arise by unequal exchange between alleles. The existence of low level germline mosaicism for new mutants further indicates that many germline mutation events are premeiotic. Physical selection of new mutants also allows minisatellite mutation rates to be estimated directly in human DNA.     

Unequal crossingover between homologous chromosomes is not the major mechanism involved in the generation of new alleles at VNTR loci

To investigate the hypothesis that unequal exchange between homologous chromosomes is involved when new alleles are generated at VNTR loci, we used genetic linkage maps to identify flanking markers surrounding a VNTR marker locus. The minisatellite probe lambda MS1 was selected, as the hypervariable locus it detects undergoes spontaneous generation of new alleles in the germline at a rate of approximately 5%. Multipoint linkage analysis placed lambda MS1 within a cluster of polymorphic marker loci on chromosome 1p. Using the two closest flanking markers, CMM8 and YNZ2, we were able to characterize 12 new-allele events in terms of crossingover between the flanking markers. Statistical analysis of these data has allowed us to reject the model that assumes that events generating new alleles always involve unequal exchange between homologous chromosomes at meiosis.     

The Yeast Pif1 Helicase Prevents Genomic Instability Caused by G-Quadruplex-Forming CEB1 Sequences In Vivo

In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Δ cells. Hence, we conclude that CEB1 instability in pif1Δ cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences.     

Mutations at the human minisatellite MS32 integrated in yeast occur with high frequency in meiosis and involve complex recombination events

Minisatellites are composed of tandem repetitive DNA sequences and are present at many positions in the human genome. They frequently mutate to new length alleles in the germline, by complex and incompletely understood recombination mechanisms which may operate during meiosis. In several minisatellites the mutation events are restricted to one end of the repeat array, indicating a possible association with elements that act in cis. Mutant alleles do not show exchange of flanking regions. To construct a model system suitable for further investigations of the mutation process, we have integrated the human minisatellite MS32, flanked by synthetic markers, in the vicinity of a meiotic recombination hot spot upstream of the LEU2 locus in the yeast Saccharomyces cerevisiae. Here we provide direct evidence for a meiotic origin of MS32 mutations. Mutation events were polarised towards both ends of the minisatellite and varied from simple duplications and deletions to complex intra- and interallelic events. Interallelic events were frequently accompanied by exchange of regions flanking the minisatellite. The results also support the notion that cis-acting elements are involved in the mutational process. The fact that MS32 mutant structures are similar in yeast and human shows that meiotic recombination plays a crucial role in both organisms and emphasises the usefulness of yeast strains harbouring minisatellites as a model system for the study of minisatellite mutation. Received: 1 March 1997 / Accepted: 16 May 1997     

Minisatellite variants generated in yeast meiosis involve DNA removal during gene conversion

Two yeast minisatellite alleles were cloned and inserted into a genetically defined interval in Saccharomyces cerevisiae. Analysis of flanking markers in combination with sequencing allowed the determination of the meiotic events that produced minisatellites with altered lengths. Tetrad analysis revealed that gene conversions, deletions, or complex combinations of both were involved in producing minisatellite variants. Similar changes were obtained following selection for nearby gene conversions or crossovers among random spores. The largest class of events involving the minisatellite was a 3:1 segregation of parental-size alleles, a class that would have been missed in all previous studies of minisatellites. Comparison of the sequences of the parental and novel alleles revealed that DNA must have been removed from the recipient array while a newly synthesized copy of donor array sequences was inserted. The length of inserted sequences did not appear to be constrained by the length of DNA that was removed. In cases where one or both sides of the insertion could be determined, the insertion endpoints were consistent with the suggestion that the event was mediated by alignment of homologous stretches of donor/recipient DNA.     

The use of synthetic tandem repeats to isolate new VNTR loci: cloning of a human hypermutable sequence.

Synthetic tandem repeats (STRs) of oligonucleotides have previously been shown to detect polymorphic loci in the human genome. Here, we report results from the use of three such probes to screen a human cosmid library. Nine of the 45 positive clones that were analyzed appear to contain highly polymorphic minisatellite or VNTR loci. The degree of enrichment for minisatellite sequences varied with the choice of STR: one provided a 15- to 20-fold enrichment (4 polymorphic loci among 10 clones), whereas 2 others gave a 3- to 5-fold enrichment (5 polymorphic probes in a total of 35 clones) compared to random screening. The 9 VNTR markers have been localized by linkage analysis in the CEPH panel and/or by in situ hybridization. Eight probes identify new loci, one of which maps to an interstitial region. One of the VNTR loci (identified by probe CEB1) was found to be hypermutable, with 52 mutation events identified among 310 children characterized in 40 CEPH families. The parental origin of the mutation could be identified in all instances, and only one mutation was found to be of maternal origin. The mutation rate in males was estimated to be approximately 15%. Segregation analysis of flanking markers suggests that mutations are not associated with crossing over. As the only previously described hypermutable minisatellite loci in humans have equal rates of male and female mutations, these observations establish that a second type of hypermutable minisatellite exists in the human genome. In neither case does the generation of new alleles appear to be associated with unequal crossing over.     

Recombination analysis of the human minisatellite MsH42 suggests the existence of two distinct pathways for initiation and resolution of recombination at MsH42 in rat testes nuclear extracts

We have previously described a GC-rich human minisatellite, termed MsH42, which exists in two allelic forms, long and short. Here, we have identified a third allele of medium length and localized the MsH42 locus in the chromosome 15q25.1 inside an intron belonging to a gene of unknown function. The recombinogenic potential of the three alleles was assayed in vitro incubating pBR322-based constructs containing two copies of the minisatellite MsH42 with its flanking sequences, in the presence of rat testes nuclear extracts. This assay system was configured to monitor only reciprocal exchange type events and not gene conversion. All MsH42 allelic sequences enhanced intramolecular homologous recombination promoting high rates (approximately 76%) of equal crossover, the long allele showing the highest recombinogenic activity. Removal of the MsH42 long allele flanking sequences, which are identical in the three alleles, provoked a decrease in the enhancement of recombination and in the frequency of equal crossovers, suggesting that these sequences are important for the recombinogenic activity and for the correct pairing between homologous sequences. The occurrence of some complex recombination events within the minisatellite MsH42 suggests the existence of processes related to polymerase slippage and unwinding with reinvasion during the repair synthesis. Our findings point toward the existence of two distinct biochemical pathways for initiation and resolution of recombination at the minisatellite MsH42. Finally, the in vitro recombination system employed in this study could provide an approach to dissect processes of repetitive DNA instability and recombination.     

A unique AT-rich hypervariable minisatellite 3' to the ApoB gene defines a high information restriction fragment length polymorphism

A DNA restriction fragment length polymorphism has been found immediately 3' to the human apoB gene. Digestion of many different human DNAs at sites flanking the region and Southern blotting analysis reveal that this region can vary in length by approximately 300 base pairs with five alleles readily distinguishable. The length polymorphism is due to a unique AT-rich minisatellite that consists primarily of a 30-base pair tandem repeat with two structurally related subunit sequences, x (ATAATTAAATATTTT) and y (ATAATTAAAATATTT). In general, the sequences repeat in an x-y order. The AT-rich region also contains variant x and y sequences that result from C or G for A substitution. Sequence analysis of one large allele revealed the expected increased number of xy repeats. In addition, similar analysis of three different smaller alleles with the same apparent size on Southern blotting analysis showed that all were of slightly different size due to minor differences in the number of xy repeats. The heterogeneity of this AT-rich minisatellite provides the basis for a highly informative restriction fragment length polymorphism of the apoB gene and should be very useful in association and linkage analysis studies of the contribution of this locus to atherosclerosis susceptibility.     

Hypermutable minisatellites,a human affair?

Minisatellites are a class of highly polymorphic GC-rich tandem repeats. They include some of the most variable loci in the human genome, with mutation rates ranging from 0.5% to >20% per generation. Structurally, they consist of 10- to 100-bp intermingled variant repeats, making them ideal tools for dissecting mechanisms of instability at tandem repeats. Distinct mutation processes generate rare intra-allelic somatic events and frequent complex conversion-like germline mutations in these repeats. Furthermore, turnover of repeats at human minisatellites is controlled by intense recombinational activity in DNA flanking the repeat array. Surprisingly, whereas other mammalian genomes possess minisatellite-like sequences, hypermutable loci have not been identified that suggest human-specific turnover processes at minisatellite arrays. Attempts to transfer minisatellite germline instability to the mouse have failed. However, yeast models are now revealing valuable information regarding the mechanisms regulating instability at these tandem repeats. Finally, minisatellites and tandem repeats provide exquisitely sensitive molecular tools to detect genomic insults such as ionizing radiation exposure. Surprisingly, by a mechanism that remains elusive, there are transgenerational increases in minisatellite instability.     

A GGCAGG motif in minisatellites affecting their germline instability

Mouse and human genomes contain hypervariable DNA regions consisting of tandem repeats of a short sequence referred to as minisatellites. This variation is thought to arise through processes such as unequal crossover or replication slippage. A mo-1 minisatellite probe comprising a 14-base pair repeat sequence reveals many polymorphic fragments even in DNA of BALB/c sublines. Oligonucleotide probes with single base substitution in the mo-1 have been synthesized and used for assessing sequence involved in generation of polymorphisms. The results indicate that the loci containing mo-1 homologues with mutation in the GGCAGG sequence are monomorphic despite the other mutants showing polymorphism. Reciprocally, locus-specific polymorphic clones, Pc-1 and Pc-2, have been isolated with hybridization to mo-1, and both are shown to contain repeated sequence comprising the GGCAGG sequence. They reveal high mutation rates of 8.8% and 3.3% per gamete, respectively. These results strongly suggest that the motif contributes to the germline instability of minisatellites.     

Meiotic interallelic conversion at the human minisatellite MS32 in yeast triggers recombination in several chromatids

Tandem repetitive DNA sequences such as minisatellites include the most polymorphic loci yet identified in the human genome. The high mutation rates at many of these loci are driven by incompletely understood recombination-based mechanisms that operate in the germline. To analyse aspects of minisatellite mutation processes and general eukaryotic recombination in meiosis that cannot be studied in humans or other mammals, including crosstalk and interplay between all four chromatids, we have previously constructed a eukaryotic model system, enabling the analysis of all four products of meiosis. In this system we have integrated alleles of the human minisatellite MS32, flanked by synthetic markers, in the vicinity of a meiotic recombination hot spot in chromosome III of Saccharomyces cerevisiae. In the present study, tetrad analysis showed that gene conversion is the predominant and possibly the universal pathway leading to interallelic transfer of repeats, with or without exchange of flanking regions. The data also suggest a hyper-recombinogenic state, triggered by interallelic mutation processes which generate a cascade of mutant alleles in the same meiosis. A number of tetrads contained identical mutant alleles of meiotic origin. Several tetrads could not be explained by the current models for minisatellite mutation. Accordingly, we here present a modified model based on the successive repair of multiple double-strand breaks.     

Cis-regulation of inter-allelic exchanges in mutation at human minisatellite MS205 in yeast.

Tandemly repeated DNA is a major component of the human genome, and includes loci contributing to human disease. Minisatellites include the most variable human loci described to date, and the mechanisms by which this variation is generated in humans have been studied in detail. Integration of human minisatellites into yeast not only provides a model for further dissecting the molecular basis of length change mutation at these loci, but also more generally allows the study of complex recombinational events in yeast. We have used human minisatellite MS205 integrated into yeast to study the structural details of length change mutations. Apart from showing that mutation at this locus in yeast has features similar to those observed at some minisatellites in humans, including meiosis-specificity, and polarity, in which exchange events are localised to one extremity of the array, we here, for the first time, directly demonstrate that a flanking element in yeast regulates the mutation process. The results therefore support the hypothesis that flanking initiators are involved in minisatellite mutation in humans. Furthermore, mutant alleles showed more complex rearrangements in one orientation than the other. The data also suggest that the mutational pathway for deletions might be different from the pathway generating inter-allelic exchanges and duplications.     

Variable germline and embryonic instability of the human minisatellite MS32 (D1S8) in transgenic mice.

Tandem repeat loci such as minisatellites and trinucleotide repeats frequently show instability. We have investigated mutation at human minisatellite MS32 (locus D1S8) transferred to transgenic mice. Three lines of hemizygous transgenic mice were studied. A single-copy line (110D) was seen to be relatively stable, whilst two multicopy lines showed structural instability of the transgene in pedigrees (lines 109 and 110A). For both these lines, mutant structures were detected as a result of mutation events having occurred in the germline or early embryo. Structural changes seen included gain or loss of minisatellite repeat units (110A and 109), alteration of DNA flanking the minisatellite repeat array (109 only) or deletion of the entire transgene (109 only). This work demonstrates that tandem repeat transgenes can show instability and thus provide additional systems for the analysis of repetitive DNA structural change in mice.     

Length Polymorphisms in Human Proline-Rich Protein Genes Generated by Intragenic Unequal Crossing over

Southern blot hybridization analysis of genomic DNAs from 44 unrelated individuals revealed extensive insertion/deletion polymorphisms within the BstNI-type loci (PRB1, PRB2, PRB3 and PRB4) of the human proline-rich protein (PRP) multigene family. Ten length variants were cloned, including alleles at each of the four PRB loci, and in every case the region of length difference was localized to the tandemly repetitious third exon. DNA sequences covering the region of length variation were determined for seven of the alleles. The data indicate (1) that the PRB loci can be divided into two subtypes, PRB1 plus PRB2, and PRB3 plus PRB4, and (2) that the length differences result from different numbers of tandem repeats in the third exons. Variant chromosomes were also identified with different numbers of PRP loci resulting from homologous but unequal exchange between the PRB1 and PRB2 loci. The overall data are compatible with the observed length variants having been generated via homologous but unequal intragenic exchange. The results also indicate that these crossover events are sensitive to the amount of homology shared between the interacting DNA strands. Allelic length variants have arisen independently at least 20 times at the PRB loci, but only one has been detected at a PRH locus. Comparison of the detailed structures of the repetitious regions in PRB and PRH loci shows that the repeats in PRB genes are very similar to each other in sequence and in length. The PRH genes contain fewer repeats, which differ considerably in their individual lengths. These differences suggest that the larger number of length variants in PRB genes is related to their greater ease of homologous but unequal pairing compared to PRH genes.     

Tetrad analysis shows that gene conversion is the major mechanism involved in mutation at the human minisatellite MS1 integrated in Saccharomyces cerevisiae

Minisatellites are arrays of tandemly repeated DNA sequences which occur at thousands of locations in the human genome. They are frequently hypervariable with respect to allele length as a result of high rates of complex and incompletely understood recombination-based germline mutation events that alter the repeat copy number. MS1 is one of the most variable minisatellites so far isolated from the human genome. We have integrated MS1, flanked by synthetic markers, in the vicinity of a hot spot for meiotic double-strand breaks upstream of the LEU2 locus in chromosome III of Saccharomyces cerevisiae. Here we present the first tetrad analysis of mutations at a human minisatellite locus. The data showed that mutant alleles occur as single mutants in one of the spores in a tetrad, also when the mutant structure was the result of a combination of intra- and inter-allelic rearrangements. The conversional transfer of repeat units from one allele to the other was associated with flanking marker conversion which always involved the same flank of the minisatellite. The results demonstrate that conversion is the predominant mechanism by which minisatellite alleles mutate to new lengths, and also support the assumption that cis-acting elements are involved in the regulation of the mutational process in humans.