Properties of natural double-strand-break sites at a recombination hotspot in Saccharomyces cerevisiae

This study addresses three questions about the properties of recombination hotspots in Saccharomyces cerevisiae: How much DNA is required for double-strand-break (DSB) site recognition? Do naturally occurring DSB sites compete with each other in meiotic recombination? What role does the sequence located at the sites of DSBs play? In S. cerevisiae, the HIS2 meiotic recombination hotspot displays a high level of gene conversion, a 3''-to-5'' conversion gradient, and two DSB sites located approximately 550 bp apart. Previous studies of hotspots, including HIS2, suggest that global chromosome structure plays a significant role in recombination activity, raising the question of how much DNA is sufficient for hotspot activity. We find that 11.5 kbp of the HIS2 region is sufficient to partially restore gene conversion and both DSBs when moved to another yeast chromosome. Using a variety of different constructs, studies of hotspots have indicated that DSB sites compete with one another for DSB formation. The two naturally occurring DSBs at HIS2 afforded us the opportunity to examine whether or not competition occurs between these native DSB sites. Small deletions of DNA at each DSB site affect only that site; analyses of these deletions show no competition occurring in cis or in trans, indicating that DSB formation at each site at HIS2 is independent. These small deletions significantly affect the frequency of DSB formation at the sites, indicating that the DNA sequence located at a DSB site can play an important role in recombination initiation.     

真核生物减数分裂重组热点的研究进展

真核生物减数分裂过程中基因组中某些区域会发生较其他区域高的重组频率,这些区域被称作减数分裂重组热点。该现象首先在酵母的研究中发现,重组区域因含有启动重组的特异位点,从而使基因组中呈现出重组不均匀分布的特征。重组热点还在真菌、玉米和人类等真核生物中发现。本文列举了不同真核生物体中具有代表性鉴别重组热点的方法,总结了目前减数分裂重组热点的研究现状,探讨了引起真核生物减数分裂交换活跃的因子和机制,并就当前存在的问题和今后发展的前景进行了讨论。     

A test of the CoHR motif associated with meiotic double-strand breaks in Saccharomyces cerevisiae

Meiotic recombination is not random along chromosomes; rather, there are preferred regions for initiation called hotspots. Although the general properties of meiotic hotspots are known, the requirements at the DNA sequence level for the determination of hotspot activity are still unclear. The sequence of six known hotspots in Saccharomyces cerevisiae was compared to identify a common homology region (CoHR). They reported that the locations of CoHR sequences correspond to mapped double-strand break (DSB) sites along three chromosomes (I, III, VI). We report here that a deletion of CoHR at HIS2, a hotspot used to identify the motif, has no significant effect on recombination. In the absence of CoHR, DSB formation occurs at a high frequency and at the same sequences as in wild-type strains. In cases where the deletion of sequences containing the CoHR motif has been shown to reduce recombination, we propose that it may be a reflection of the location of the deletion, rather than the loss of CoHR, per se.     

Genetic Crossovers Are Predicted Accurately by the Computed Human Recombination Map

Hotspots of meiotic recombination can change rapidly over time. This instability and the reported high level of inter-individual variation in meiotic recombination puts in question the accuracy of the calculated hotspot map, which is based on the summation of past genetic crossovers. To estimate the accuracy of the computed recombination rate map, we have mapped genetic crossovers to a median resolution of 70 Kb in 10 CEPH pedigrees. We then compared the positions of crossovers with the hotspots computed from HapMap data and performed extensive computer simulations to compare the observed distributions of crossovers with the distributions expected from the calculated recombination rate maps. Here we show that a population-averaged hotspot map computed from linkage disequilibrium data predicts well present-day genetic crossovers. We find that computed hotspot maps accurately estimate both the strength and the position of meiotic hotspots. An in-depth examination of not-predicted crossovers shows that they are preferentially located in regions where hotspots are found in other populations. In summary, we find that by combining several computed population-specific maps we can capture the variation in individual hotspots to generate a hotspot map that can predict almost all present-day genetic crossovers.     

No dosage effect of recombinational hotspots in the mouse major histocompatibility complex

The sites of meiotic recombination in the proximal region of the mouse major histocompatibility complex (MHC) are clustered at hotspots. Some MHC haplotypes derived from Asian wild mice increase the frequency of recombination at such hotspots when heterozygous with standard laboratory haplotypes. The wm7 and cas3 haplotypes, have a hotspot close to the Lmp-2 gene (Lmp-2 hotspot), and the cas4 haplotype has a hotspot about 100 kilobase (kb) proximal, close to the Pb gene (Pb hotspot). To examine the effect of a double dose of hotspots, we estimated the rate of recombination and determined the location of the breakpoints in crosses of wm7/cas3 and wm7/cas4. In 3570 backcross progeny we identified 29 new recombinants in the H-2K to Ab interval, at a frequency of 0.81%. This frequency is 40-fold higher than in crosses between laboratory haplotypes and very similar to those previously obtained in crosses between these wild and standard laboratory haplotypes. Thus, a double dose of hotspots has no additive effect on the frequency of meiotic recombination. The site-specificity of recombination was also conserved. Twenty-three breakpoints were confined within 5.4 kb in the Lmp-2 hotspot, and six breakpoints from the cas4 cross were located in the Pb hotspot, which we have now confined to a 15 kb segment. Correspondence to: T. Shiroishi.     

A combination of cis and trans control can solve the hotspot conversion paradox

There is growing evidence that in a variety of organisms the majority of meiotic recombination events occur at a relatively small fraction of loci, known as recombination hotspots. If hotspot activity results from the DNA sequence at or near the hotspot itself (in cis), these hotspots are expected to be rapidly lost due to biased gene conversion, unless there is strong selection in favor of the hotspot itself. This phenomenon makes it very difficult to maintain existing hotspots and even more difficult for new hotspots to evolve; it has therefore come to be known as the "hotspot conversion paradox." I develop an analytical framework for exploring the evolution of recombination hotspots under the forces of selection, mutation, and conversion. I derive the general conditions under which cis- and trans-controlled hotspots can be maintained, as well as those under which new hotspots controlled by both a cis and a trans locus can invade a population. I show that the conditions for maintenance of and invasion by trans- or cis-plus-trans-controlled hotspots are broader than for those controlled entirely in cis. Finally, I show that a combination of cis and trans control may allow for long-lived polymorphisms in hotspot activity, the patterns of which may explain some recently observed features of recombination hotspots.     

The Red Queen theory of recombination hotspots

Recombination hotspots are small chromosomal regions, where meiotic crossover events happen with high frequency. Recombination is initiated by a double‐strand break (DSB) that requires the intervention of the molecular repair mechanism. The DSB repair mechanism may result in the exchange of homologous chromosomes (crossover) and the conversion of the allelic sequence that breaks into the one that does not break (biased gene conversion). Biased gene conversion results in a transmission advantage for the allele that does not break, thus preventing recombination and rendering recombination hotspots transient. How is it possible that recombination hotspots persist over evolutionary time (maintaining the average chromosomal crossover rate) when they are self‐destructive? This fundamental question is known as the recombination hotspot paradox and has attracted much attention in recent years. Yet, that attention has not translated into a fully satisfactory answer. No existing model adequately explains all aspects of the recombination hotspot paradox. Here, we formulate an intragenomic conflict model resulting in Red Queen dynamics that fully accounts for all empirical observations regarding the molecular mechanisms of recombination hotspots, the nonrandom targeting of the recombination machinery to hotspots and the evolutionary dynamics of hotspot turnover.     

Live hot, die young: transmission distortion in recombination hotspots

There is strong evidence that hotspots of meiotic recombination in humans are transient features of the genome. For example, hotspot locations are not shared between human and chimpanzee. Biased gene conversion in favor of alleles that locally disrupt hotspots is a possible explanation of the short lifespan of hotspots. We investigate the implications of such a bias on human hotspots and their evolution. Our results demonstrate that gene conversion bias is a sufficiently strong force to produce the observed lack of sharing of intense hotspots between species, although sharing may be much more common for weaker hotspots. We investigate models of how hotspots arise, and find that only models in which hotspot alleles do not initially experience drive are consistent with observations of rather hot hotspots in the human genome. Mutations acting against drive cannot successfully introduce such hotspots into the population, even if there is direct selection for higher recombination rates, such as to ensure correct segregation during meiosis. We explore the impact of hotspot alleles on patterns of haplotype variation, and show that such alleles mask their presence in population genetic data, making them difficult to detect.     

The M26 hotspot of Schizosaccharomyces pombe stimulates meiotic ectopic recombination and chromosomal rearrangements.

Homologous recombination is increased during meiosis between DNA sequences at the same chromosomal position (allelic recombination) and at different chromosomal positions (ectopic recombination). Recombination hotspots are important elements in controlling meiotic allelic recombination. We have used artificially dispersed copies of the ade6 gene in Schizosaccharomyces pombe to study hotspot activity in meiotic ectopic recombination. Ectopic recombination was reduced 10-1000-fold relative to allelic recombination, and was similar to the low frequency of ectopic recombination between naturally repeated sequences in S. pombe. The M26 hotspot was active in ectopic recombination in some, but not all, integration sites, with the same pattern of activity and inactivity in ectopic and allelic recombination. Crossing over in ectopic recombination, resulting in chromosomal rearrangements, was associated with 35-60% of recombination events and was stimulated 12-fold by M26. These results suggest overlap in the mechanisms of ectopic and allelic recombination and indicate that hotspots can stimulate chromosomal rearrangements.     

Distinct histone modifications define initiation and repair of meiotic recombination in the mouse

Little is known about the factors determining the location and activity of the rapidly evolving meiotic crossover hotspots that shape genome diversity. Here, we show that several histone modifications are enriched at the active mouse Psmb9 hotspot, and we distinguish those marks that precede from those that follow hotspot recombinational activity. H3K4Me3, H3K4Me2 and H3K9Ac are specifically enriched in the chromatids that carry an active initiation site, and in the absence of DNA double-strand breaks (DSBs) in Spo11^−/− mice. We thus propose that these marks are part of the substrate for recombination initiation at the Psmb9 hotspot. In contrast, hyperacetylation of H4 is increased as a consequence of DSB formation, as shown by its dependency on Spo11 and by the enrichment detected on both recombining chromatids. In addition, the comparison with another hotspot, Hlx1, strongly suggests that H3K4Me3 and H4 hyperacetylation are common features of DSB formation and repair, respectively. Altogether, the chromatin signatures of the Psmb9 and Hlx1 hotspots provide a basis for understanding the distribution of meiotic recombination.     

Persistence and loss of meiotic recombination hotspots

The contradiction between the long-term persistence of the chromosomal hotspots that initiate meiotic recombination and the self-destructive mechanism by which they act strongly suggests that our understanding of recombination is incomplete. This "hotspot paradox" has been reinforced by the finding that biased gene conversion also removes active hotspots from human sperm. To investigate the requirements for hotspot persistence, we developed a detailed computer simulation model of their activity and its evolutionary consequences. With this model, unopposed hotspot activity could drive strong hotspots from 50% representation to extinction within 70 generations. Although the crossing over that hotspots cause can increase population fitness, this benefit was always too small to slow the loss of hotspots. Hotspots could not be maintained by plausible rates of de novo mutation, nor by crossover interference, which alters the frequency and/or spacing of crossovers. Competition among hotspots for activity-limiting factors also did not prevent their extinction, although the rate of hotspot loss was slowed. Key factors were the probability that the initiating hotspot allele is destroyed and the nonmeiotic contributions hotspots make to fitness. Experimental investigation of these deserves high priority, because until the paradox is resolved all components of the mechanism are open to doubt.     

The Tn3 β-Lactamase Gene Acts as a Hotspot for Meiotic Recombination in Yeast

Although genetic distances are often assumed to be proportional to physical distances, chromosomal regions with unusually high (hotspots) or low (coldspots) levels of meiotic recombination have been described in a number of genetic systems. In general, the DNA sequences responsible for these effects have not been determined. We report that the 5' region of the beta-lactamase (ampR) gene of the bacterial transposon Tn3 is a hotspot for meiotic recombination when inserted into the chromosomes of the yeast Saccharomyces cerevisiae. When these sequences are homozygous, both crossing over and gene conversion are locally stimulated. The 5' end of the beta-lactamase gene is about 100-fold "hotter" for crossovers than an average yeast DNA sequence.     

Enrichment of meiotic recombination hotspot sequences by avidin capture technology

About 40% of the hotspots for meiotic recombination contain the degenerate consensus sequence 5′-CCNCCNTNNCCNC-3′. Here we present a novel protocol for enriching hotspot sequences from digested genomic DNA by using biotinylated oligonucleotides and streptavidin-coated magnetic beads. The captured hotspots can be released by simple digestion with restriction enzymes for subsequent characterization by second generation sequencing or PCR. The capture protocol specifically enriches hotspot sequences, judged by using fluorophore-conjugated synthetic oligonucleotides and synthetic double-stranded oligonucleotides in combination with PCR. The capture protocol enriches single-stranded DNA, denatured double-stranded DNA, and large fragments (> 3000 bp) of digested plasmid DNA with good efficacy. No false positive and false negatives were detected when enriching digested DNA from human cell cultures and primary human cells. The protocol can probably be adapted to enriching sequences other than the hotspot sequence by altering the sequence in the capture oligonucleotide. We intend to apply this protocol in studies assessing effects of micronutrient status on meiotic recombination events in human sperm.     

A Highlights from MBoC Selection: EWSR1 affects PRDM9-dependent histone 3 methylation and provides a link between recombination hotspots and the chromosome axis protein REC8

Meiotic recombination in most mammals requires recombination hotspot activation through the action of the histone 3 Lys-4 and Lys-36 methyltransferase PRDM9 to ensure successful double-strand-break initiation and repair. Here we show that EWSR1, a protein whose role in meiosis was not previously clarified in detail, binds to both PRDM9 and pREC8, a phosphorylated meiosis-specific cohesin, in male meiotic cells. We created a Ewsr1 conditional knockout mouse model to deplete EWSR1 before the onset of meiosis and found that absence of EWSR1 causes meiotic arrest with decreased histone trimethylation at meiotic hotspots, impaired DNA double-strand-break repair, and reduced crossover number. Our results demonstrate that EWSR1 is essential for promoting PRDM9-dependent histone methylation and normal meiotic progress, possibly by facilitating the linking between PRDM9-bound hotspots and the nascent chromosome axis through its component cohesin pREC8.     

Cis- and trans-acting elements regulate the mouse Psmb9 meiotic recombination hotspot

In most eukaryotes, the prophase of the first meiotic division is characterized by a high level of homologous recombination between homologous chromosomes. Recombination events are not distributed evenly within the genome, but vary both locally and at large scale. Locally, most recombination events are clustered in short intervals (a few kilobases) called hotspots, separated by large intervening regions with no or very little recombination. Despite the importance of regulating both the frequency and the distribution of recombination events, the genetic factors controlling the activity of the recombination hotspots in mammals are still poorly understood. We previously characterized a recombination hotspot located close to the Psmb9 gene in the mouse major histocompatibility complex by sperm typing, demonstrating that it is a site of recombination initiation. With the goal of uncovering some of the genetic factors controlling the activity of this initiation site, we analyzed this hotspot in both male and female germ lines and compared the level of recombination in different hybrid mice. We show that a haplotype-specific element acts at distance and in trans to activate about 2,000-fold the recombination activity at Psmb9. Another haplotype-specific element acts in cis to repress initiation of recombination, and we propose this control to be due to polymorphisms located within the initiation zone. In addition, we describe subtle variations in the frequency and distribution of recombination events related to strain and sex differences. These findings show that most regulations observed act at the level of initiation and provide the first analysis of the control of the activity of a meiotic recombination hotspot in the mouse genome that reveals the interactions of elements located both in and outside the hotspot.     

DNA sequence-mediated, evolutionarily rapid redistribution of meiotic recombination hotspots

Hotspots regulate the position and frequency of Spo11 (Rec12)-initiated meiotic recombination, but paradoxically they are suicidal and are somehow resurrected elsewhere in the genome. After the DNA sequence-dependent activation of hotspots was discovered in fission yeast, nearly two decades elapsed before the key realizations that (A) DNA site-dependent regulation is broadly conserved and (B) individual eukaryotes have multiple different DNA sequence motifs that activate hotspots. From our perspective, such findings provide a conceptually straightforward solution to the hotspot paradox and can explain other, seemingly complex features of meiotic recombination. We describe how a small number of single-base-pair substitutions can generate hotspots de novo and dramatically alter their distribution in the genome. This model also shows how equilibrium rate kinetics could maintain the presence of hotspots over evolutionary timescales, without strong selective pressures invoked previously, and explains why hotspots localize preferentially to intergenic regions and introns. The model is robust enough to account for all hotspots of humans and chimpanzees repositioned since their divergence from the latest common ancestor.