Mapping candidate hotspots of meiotic recombination in segments of human DNA cloned in the yeast<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The hotspots of meiotic recombination in the human genome can be localized by genetic techniques. The resolution of these techniques is in the range of kilobases and depends on the density of the physical markers identifying allelic variants of the chromosomal loci. We thought it would be interesting to localize these sites with higher resolution. Assuming that some human chromosomal sites conserve their propensity for recombination when cloned in yeast, we localized the hotspots of recombination in several yeast artificial chromosomes (YACs) carrying human DNA. A number of potential recombination hotspots could be identified in the clones studied. Among them there are two classes of sites that are particularly recombination prone also in human meiotic cells: sites associated with CpG islands and sites located in the vicinity of long minisatellite sequences.Communicated by G. P. Georgiev     

A discrete class of intergenic DNA dictates meiotic DNA break hotspots in fission yeast

Meiotic recombination is initiated by DNA double-strand breaks (DSBs) made by Spo11 (Rec12 in fission yeast), which becomes covalently linked to the DSB ends. Like recombination events, DSBs occur at hotspots in the genome, but the genetic factors responsible for most hotspots have remained elusive. Here we describe in fission yeast the genome-wide distribution of meiosis-specific Rec12-DNA linkages, which closely parallel DSBs measured by conventional Southern blot hybridization. Prominent DSB hotspots are located ~65 kb apart, separated by intervals with little or no detectable breakage. Most hotspots lie within exceptionally large intergenic regions. Thus, the chromosomal architecture responsible for hotspots in fission yeast is markedly different from that of budding yeast, in which DSB hotspots are much more closely spaced and, in many regions of the genome, occur at each promoter. Our analysis in fission yeast reveals a clearly identifiable chromosomal feature that can predict the majority of recombination hotspots across a whole genome and provides a basis for searching for the chromosomal features that dictate hotspots of meiotic recombination in other organisms, including humans.     

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。     

Differential Relationship of DNA Replication Timing to Different Forms of Human Mutation and Variation

Human genetic variation is distributed nonrandomly across the genome, though the principles governing its distribution are only partially known. DNA replication creates opportunities for mutation, and the timing of DNA replication correlates with the density of SNPs across the human genome. To enable deeper investigation of how DNA replication timing relates to human mutation and variation, we generated a high-resolution map of the human genome’s replication timing program and analyzed its relationship to point mutations, copy number variations, and the meiotic recombination hotspots utilized by males and females. DNA replication timing associated with point mutations far more strongly than predicted from earlier analyses and showed a stronger relationship to transversion than transition mutations. Structural mutations arising from recombination-based mechanisms and recombination hotspots used more extensively by females were enriched in early-replicating parts of the genome, though these relationships appeared to relate more strongly to the genomic distribution of causative sequence features. These results indicate differential and sex-specific relationship of DNA replication timing to different forms of mutation and recombination.     

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.     

Molecular mechanisms for environmentally induced and evolutionarily rapid redistribution (plasticity) of meiotic recombination

It has long been known (circa 1917) that environmental conditions, as well as speciation, can affect dramatically the frequency distribution of Spo11/Rec12-dependent meiotic recombination. Here, by analyzing DNA sequence-dependent meiotic recombination hotspots in the fission yeast Schizosaccharomyces pombe, we reveal a molecular basis for these phenomena. The impacts of changing environmental conditions (temperature, nutrients, and osmolarity) on local rates of recombination are mediated directly by DNA site-dependent hotspots (M26, CCAAT, and Oligo-C). This control is exerted through environmental condition-responsive signal transduction networks (involving Atf1, Pcr1, Php2, Php3, Php5, and Rst2). Strikingly, individual hotspots modulate rates of recombination over a very broad dynamic range in response to changing conditions. They can range from being quiescent to being highly proficient at promoting activity of the basal recombination machinery (Spo11/Rec12 complex). Moreover, each different class of hotspot functions as an independently controlled rheostat; a condition that increases the activity of one class can decrease the activity of another class. Together, the independent modulation of recombination rates by each different class of DNA site-dependent hotspots (of which there are many) provides a molecular mechanism for highly dynamic, large-scale changes in the global frequency distribution of meiotic recombination. Because hotspot-activating DNA sites discovered in fission yeast are conserved functionally in other species, this process can also explain the previously enigmatic, Prdm9-independent, evolutionarily rapid changes in hotspot usage between closely related species, subspecies, and isolated populations of the same species.     

Elevated Mutagenicity in Meiosis and Its Mechanism

Diploid germ cells produce haploid gametes through meiosis, a unique type of cell division. Independent reassortment of parental chromosomes and their recombination leads to ample genetic variability among the gametes. Importantly, new mutations also occur during meiosis, at frequencies much higher than during the mitotic cell cycles. These meiotic mutations are associated with genetic recombination and depend on double‐strand breaks (DSBs) that initiate crossing over. Indeed, sequence variation among related strains is greater around recombination hotspots than elsewhere in the genome, presumably resulting from recombination‐associated mutations. Significantly, enhanced mutagenicity in meiosis may lead to faster divergence during evolution, as germ‐line mutations are the ones that are transmitted to the progeny and thus have an evolutionary impact. The molecular basis for mutagenicity in meiosis may be related to the repair of meiotic DSBs by polymerases, or to the exposure of single‐strand DNA to mutagenic agents during its repair.     

New and old ways to control meiotic recombination

The unique segregation of homologs, rather than sister chromatids, at the first meiotic division requires the formation of crossovers (COs) between homologs by meiotic recombination in most species. Crossovers do not form at random along chromosomes. Rather, their formation is carefully controlled, both at the stage of formation of DNA double-strand breaks (DSBs) that can initiate COs and during the repair of these DSBs. Here, we review control of DSB formation and two recently recognized controls of DSB repair: CO homeostasis and CO invariance. Crossover homeostasis maintains a constant number of COs per cell when the total number of DSBs in a cell is experimentally or stochastically reduced. Crossover invariance maintains a constant CO density (COs per kb of DNA) across much of the genome despite strong DSB hotspots in some intervals. These recently uncovered phenomena show that CO control is even more complex than previously suspected.     

Chromatin accessibility shapes meiotic recombination in mouse primordial germ cells through assisting double-strand breaks and loop formation

Meiotic recombination is a driver of evolution, and aberrant recombination is a major contributor to aneuploidy in mammals. Mechanism of recombination remains elusive yet. Here, we present a computational analysis to explore recombination-related dynamics of chromatin accessibility in mouse primordial germ cells (PGCs). Our data reveals that: (1) recombination hotspots which get accessible at meiosis-specific DNase I-hypersensitive sites (DHSs) only when PGCs enter meiosis are located preferentially in intronic and distal intergenic regions; (2) stable DHSs maintained stably across PGC differentiation are enriched by CTCF motifs and CTCF binding and mediate chromatin loop formation; (3) compared with the specific DHSs aroused at meiotic stage, stable DHSs are largely encoded in DNA sequence and also enriched by epigenetic marks; (4) PRDM9 is likely to target nucleosome-occupied hotspot regions and remodels local chromatin structure to make them accessible for recombination machinery; and (5) cells undergoing meiotic recombination are deficient in TAD structure and chromatin loop arrays are organized regularly along the axis formed between homologous chromosomes. Taken together, by analyzing DHS-related DNA features, epigenetic marks and 3D genome structure, we revealed some specific roles of chromatin accessibility in recombination, which would expand our understanding of recombination mechanism.     

Recombination hotspots: Models and tools for detection

Recombination hotspots are the regions within the genome where the rate, and the frequency of recombination are optimum with a size varying from 1 to 2 kb. The recombination event is mediated by the double-stranded break formation, guided by the combined enzymatic action of DNA topoisomerase and Spo 11 endonuclease. These regions are distributed non-uniformly throughout the human genome and cause distortions in the genetic map. Numerous lines of evidence suggest that the number of hotspots known in humans has increased manifold in recent years. A few facts about the hotspot evolutions were also put forward, indicating the differences in the hotspot position between chimpanzees and humans. In mice, recombination hot spots were found to be clustered within the major histocompatibility complex (MHC) region. Several models, that help explain meiotic recombination has been proposed. Moreover, scientists also developed some computational tools to locate the hotspot position and estimate their recombination rate in humans is of great interest to population and medical geneticists. Here we reviewed the molecular mechanisms, models and in silico prediction techniques of hot spot residues.     

DNA double-strand breaks in meiosis: Checking their formation, processing and repair

DNA double-strand breaks (DSBs) are highly hazardous for genome integrity, but meiotic cells deliberately introduce them into their genome in order to initiate homologous recombination, which ensures proper homologous chromosome segregation. To minimize the risk of deleterious effects, meiotic DSB formation, processing and repair are tightly regulated in order to occur only at the right time and place. Furthermore, a highly conserved signal-transduction pathway, called meiotic recombination checkpoint, coordinates DSB repair with meiotic progression and promotes meiotic recombination.     

Distribution of Recombination Hotspots in the Human Genome – A Comparison of Computer Simulations with Real Data

Recombination is the main cause of genetic diversity. Thus, errors in this process can lead to chromosomal abnormalities. Recombination events are confined to narrow chromosome regions called hotspots in which characteristic DNA motifs are found. Genomic analyses have shown that both recombination hotspots and DNA motifs are distributed unevenly along human chromosomes and are much more frequent in the subtelomeric regions of chromosomes than in their central parts. Clusters of motifs roughly follow the distribution of recombination hotspots whereas single motifs show a negative correlation with the hotspot distribution. To model the phenomena related to recombination, we carried out computer Monte Carlo simulations of genome evolution. Computer simulations generated uneven distribution of hotspots with their domination in the subtelomeric regions of chromosomes. They also revealed that purifying selection eliminating defective alleles is strong enough to cause such hotspot distribution. After sufficiently long time of simulations, the structure of chromosomes reached a dynamic equilibrium, in which number and global distribution of both hotspots and defective alleles remained statistically unchanged, while their precise positions were shifted. This resembles the dynamic structure of human and chimpanzee genomes, where hotspots change their exact locations but the global distributions of recombination events are very similar.     

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.     

Branching out: meiotic recombination and its regulation

Homologous recombination is a dynamic process by which DNA sequences and strands are exchanged. In meiosis, the reciprocal DNA recombination events called crossovers are central to the generation of genetic diversity in gametes and are required for homolog segregation in most organisms. Recent studies have shed light on how meiotic crossovers and other recombination products form, how their position and number are regulated and how the DNA molecules undergoing recombination are chosen. These studies indicate that the long-dominant, unifying model of recombination proposed by Szostak et al. applies, with modification, only to a subset of recombination events. Instead, crossover formation and its control involve multiple pathways, with considerable variation among model organisms. These observations force us to 'branch out' in our thinking about meiotic recombination.     

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.     

Transmission Distortion Affecting Human Noncrossover but Not Crossover Recombination: A Hidden Source of Meiotic Drive

Meiotic recombination ensures the correct segregation of homologous chromosomes during gamete formation and contributes to DNA diversity through both large-scale reciprocal crossovers and very localised gene conversion events, also known as noncrossovers. Considerable progress has been made in understanding factors such as PRDM9 and SNP variants that influence the initiation of recombination at human hotspots but very little is known about factors acting downstream. To address this, we simultaneously analysed both types of recombinant molecule in sperm DNA at six highly active hotspots, and looked for disparity in the transmission of allelic variants indicative of any cis-acting influences. At two of the hotspots we identified a novel form of biased transmission that was exclusive to the noncrossover class of recombinant, and which presumably arises through differences between crossovers and noncrossovers in heteroduplex formation and biased mismatch repair. This form of biased gene conversion is not predicted to influence hotspot activity as previously noted for SNPs that affect recombination initiation, but does constitute a powerful and previously undetected source of recombination-driven meiotic drive that by extrapolation may affect thousands of recombination hotspots throughout the human genome. Intriguingly, at both of the hotspots described here, this drive favours strong (G/C) over weak (A/T) base pairs as might be predicted from the well-established correlations between high GC content and recombination activity in mammalian genomes.     

Surveillance mechanisms monitoring chromosome breaks during mitosis and meiosis

DNA double-strand breaks (DSBs) are highly hazardous for genome integrity, because failure to repair them can lead to genome rearrangements or chromosome loss. They can arise at unpredictable locations as a consequence of DNA damage during both the mitotic and the meiotic cell cycle or in a programmed manner during meiosis. Cellular response to accidental or programmed DSBs involves highly conserved surveillance mechanisms, called DNA damage checkpoint and recombination checkpoint, which coordinate DSB repair with mitotic or meiotic cell cycle progression, respectively. Although these protective signal-transduction pathways share several upstream components, activation of the recombination checkpoint requires meiosis-specific proteins. These proteins are structural components of the meiotic chromosomes, indicating that the system monitoring programmed meiotic DSBs is an integral part of the chromosome structure formed during meiosis.     

Competition between Adjacent Meiotic Recombination Hotspots in the Yeast Saccharomyces Cerevisiae

In a wild-type strain of Saccharomyces cerevisiae, a hotspot for meiotic recombination is located upstream of the HIS4 gene. An insertion of a 49-bp telomeric sequence into the coding region of HIS4 strongly stimulates meiotic recombination and the local formation of meiosis-specific double-strand DNA breaks (DSBs). When strains are constructed in which both hotspots are heterozygous, hotspot activity is substantially less when the hotspots are on the same chromosome than when they are on opposite chromosomes.     

Recombination correlates with synaptonemal complex length and chromatin loop size in bovids—insights into mammalian meiotic chromosomal organization

Homologous chromosomes exchange genetic information through recombination during meiosis, a process that increases genetic diversity, and is fundamental to sexual reproduction. In an attempt to shed light on the dynamics of mammalian recombination and its implications for genome organization, we have studied the recombination characteristics of 112 individuals belonging to 28 different species in the family Bovidae. In particular, we analyzed the distribution of RAD51 and MLH1 foci during the meiotic prophase I that serve, respectively, as proxies for double-strand breaks (DSBs) which form in early stages of meiosis and for crossovers. In addition, synaptonemal complex length and meiotic DNA loop size were estimated to explore how genome organization determines DSBs and crossover patterns. We show that although the number of meiotic DSBs per cell and recombination rates observed vary between individuals of the same species, these are correlated with diploid number as well as with synaptonemal complex and DNA loop sizes. Our results illustrate that genome packaging, DSB frequencies, and crossover rates tend to be correlated, while meiotic chromosomal axis length and DNA loop size are inversely correlated in mammals. Moreover, axis length, DSB frequency, and crossover frequencies all covary, suggesting that these correlations are established in the early stages of meiosis.