Meiotic recombination hot spots and human DNA diversity

Meiotic recombination plays a key role in the maintenance of sequence diversity in the human genome. However, little is known about the fine-scale distribution and processes of recombination in human chromosomes, or how these impact on patterns of human diversity. We have therefore developed sperm typing systems that allow human recombination to be analysed at very high resolution. The emerging picture is that human crossovers are far from randomly distributed but instead are targeted into very narrow hot spots that can profoundly influence patterns of haplotype diversity in the human genome. These hot spots provide fundamental information on processes of human crossover and gene conversion, as well as evidence that they can violate basic rules of Mendelian inheritance.     

Meiotic recombination hot spots and cold spots

Meiotic recombination events are distributed unevenly throughout eukaryotic genomes. This inhomogeneity leads to distortions of genetic maps that can hinder the ability of geneticists to identify genes by map-based techniques. Various lines of evidence, particularly from studies of yeast, indicate that the distribution of recombination events might reflect, at least in part, global features of chromosome structure, such as the distribution of modified nucleosomes.     

Identifying and retargeting transcriptional hot spots in the human genome

Mammalian cell line development requires streamlined methodologies that will reduce both the cost and time to identify candidate cell lines. Improvements in site‐specific genomic editing techniques can result in flexible, predictable, and robust cell line engineering. However, an outstanding question in the field is the specific site of integration. Here, we seek to identify productive loci within the human genome that will result in stable, high expression of heterologous DNA. Using an unbiased, random integration approach and a green fluorescent reporter construct, we identify ten single‐integrant, recombinant human cell lines that exhibit stable, high‐level expression. From these cell lines, eight unique corresponding integration loci were identified. These loci are concentrated in non‐protein coding regions or intronic regions of protein coding genes. Expression mapping of the surrounding genes reveals minimal disruption of endogenous gene expression. Finally, we demonstrate that targeted de novo integration at one of the identified loci, the 12^th exon‐intron region of the GRIK1 gene on chromosome 21, results in superior expression and stability compared to the standard, illegitimate integration approach at levels approaching 4‐fold. The information identified here along with recent advances in site‐specific genomic editing techniques can lead to expedited cell line development.     

Molecular features of meiotic recombination hot spots

Meiotic recombination occurs preferentially at certain regions called hot spots and is important for generating genetic diversity and proper segregation of chromosomes during meiosis. Hot spots have been characterized most extensively in yeast, mice and humans. The development of methods based on sperm typing and population genetics has facilitated rapid and high-resolution mapping of hot spots in mice and humans in recent years. With increasing information becoming available on meiotic recombination in different species, it is now possible to compare several molecular features associated with hot-spot loci. Further, there have been advances in our knowledge of the factors influencing hot-spot activity and the role that they play in structuring the genome into haplotype blocks. We review the molecular features associated with hot spots in terms of their properties and mechanisms underlying their function and distribution. A large number of these features seem to be shared among hot spots from different species suggesting common mechanisms for their formation and function.     

Natural meiotic recombination hot spots in the Schizosaccharomyces pombe genome successfully predicted from the simple sequence motif M26

The M26 hot spot of meiotic recombination in Schizosaccharomyces pombe is the eukaryotic hot spot most thoroughly investigated at the nucleotide level. The minimum sequence required for M26 activity was previously determined to be 5'-ATGACGT-3'. Originally identified by a mutant allele, ade6-M26, the M26 heptamer sequence occurs in the wild-type S. pombe genome approximately 300 times, but it has been unclear whether any of these are active hot spots. Recently, we showed that the M26 heptamer forms part of a larger consensus sequence, which is significantly more active than the heptamer alone. We used this expanded sequence as a guide to identify a smaller number of sites most likely to be active hot spots. Ten of the 15 sites tested showed meiotic DNA breaks, a hallmark of recombination hot spots, within 1 kb of the M26 sequence. Among those 10 sites, one occurred within a gene, cds1(+), and hot spot activity of this site was confirmed genetically. These results are, to our knowledge, the first demonstration in any organism of a simple, defined nucleotide sequence accurately predicting the locations of natural meiotic recombination hot spots. M26 may be the first example among a diverse group of simple sequences that determine the distribution, and hence predictability, of meiotic recombination hot spots in eukaryotic genomes.     

Distinct functions of MLH3 at recombination hot spots in the mouse

The four mammalian MutL homologs (MLH1, MLH3, PMS1, and PMS2) participate in a variety of events, including postreplicative DNA repair, prevention of homeologous recombination, and crossover formation during meiosis. In this latter role, MLH1-MLH3 heterodimers predominate and are essential for prophase I progression. Previous studies demonstrated that mice lacking Mlh1 exhibit a 90% reduction in crossing over at the Psmb9 hot spot while noncrossovers, which do not result in exchange of flanking markers but arise from the same double-strand break event, are unaffected. Using a PCR-based strategy that allows for detailed analysis of crossovers and noncrossovers, we show here that Mlh3(-/-) exhibit a 85-94% reduction in the number of crossovers at the Psmb9 hot spot. Most of the remaining crossovers in Mlh3(-/-) meiocytes represent simple exchanges similar to those seen in wild-type mice, with a small fraction (6%) representing complex events that can extend far from the initiation zone. Interestingly, we detect an increase of noncrossovers in Mlh3(-/-) spermatocytes. These results suggest that MLH3 functions predominantly with MLH1 to promote crossovers, while noncrossover events do not require these activities. Furthermore, these results indicate that approximately 10% of crossovers in the mouse are independent of MLH3, suggesting the existence of alternative crossover pathways in mammals.     

A multigene deletion in the human IGH constant region locus involves highly homologous hot spots of recombination

A simultaneous absence of the IgG1, IgG2, IgG4, and IgA1 immunoglobulins (Ig) was unambiguously demonstrated in six healthy individuals of two different families (family HASS and family TOU). These individuals were shown to be homozygous for a large deletion in the immunoglobulin heavy chain constant region locus. This deletion, which encompasses the G1-EP1-A1-GP-G2-G4 genes, allowed us to predict an order for the IgCH genes and to localize GP between A1 and G2. In this paper, we study the deletion-recombination point in the IGH locus of individual EZZ from the TOU family. We show that the distance between the G3 and the E genes on the EZZ recombinant chromosome is 24.7 kb and that the multigene deletion in the IgCH locus involves two highly homologous regions (hsg3 and hsg4) which are hot spots of recombination, outside of the switch sequences.     

线粒体微卫星DNA不稳定性与肿瘤关系的研究进展

线粒体DNA直接控制细胞氧化磷酸化过程,其基因组序列发生突变尤其是微卫星DNA 不稳定性会严重降低线粒体产生能量的功能,诱发细胞程序性死亡和癌细胞形成。目前,有关人类线粒体微卫星DNA不稳定性与肿瘤关系的研究越来越受到关注。仅就微卫星DNA的特点和不稳定性形成的机理以及与肿瘤关系的研究进展作以概述。     

Linkage disequilibrium and inference of ancestral recombination in 538 single-nucleotide polymorphism clusters across the human genome

The prospect of using linkage disequilibrium (LD) for fine-scale mapping in humans has attracted considerable attention, and, during the validation of a set of single-nucleotide polymorphisms (SNPs) for linkage analysis, a set of data for 4,833 SNPs in 538 clusters was produced that provides a rich picture of local attributes of LD across the genome. LD estimates may be biased depending on the means by which SNPs are first identified, and a particular problem of ascertainment bias arises when SNPs identified in small heterogeneous panels are subsequently typed in larger population samples. Understanding and correcting ascertainment bias is essential for a useful quantitative assessment of the landscape of LD across the human genome. Heterogeneity in the population recombination rate, rho=4Nr, along the genome reflects how variable the density of markers will have to be for optimal coverage. We find that ascertainment-corrected rho varies along the genome by more than two orders of magnitude, implying great differences in the recombinational history of different portions of our genome. The distribution of rho is unimodal, and we show that this is compatible with a wide range of mixtures of hotspots in a background of variable recombination rate. Although rho is significantly correlated across the three population samples, some regions of the genome exhibit population-specific spikes or troughs in rho that are too large to be explained by sampling. This result is consistent with differences in the genealogical depth of local genomic regions, a finding that has direct bearing on the design and utility of LD mapping and on the National Institutes of Health HapMap project.     

Increased recombination intermediates and homologous integration hot spots at DNA replication origins

We have studied the relationship between DNA replication and recombination in Schizosaccharomyces pombe using two-dimensional gel electrophoresis and functional analysis. Our results indicate that the activation of replication origins (ORIs) during the mitotic cell cycle is associated with the generation of joint DNA molecules between sister chromatids. The frequency of integration by homologous recombination was up to 50-fold higher than the genomic average within a narrow window overlapping the ars1 replication initiation site. The S. pombe rad22Delta, rhp51Delta, and rhp54Delta mutants, deficient in mitotic recombination, activate ORIs very inefficiently and accumulate abnormal replication intermediates. These results focus on the general link between replication and recombination previously found in several systems and suggest a role for recombination in the initiation of eukaryotic DNA replication.     

The relationship between domain duplication and recombination

Protein domains represent the basic evolutionary units that form proteins. Domain duplication and shuffling by recombination are probably the most important forces driving protein evolution and hence the complexity of the proteome. While the duplication of whole genes as well as domain-encoding exons increases the abundance of domains in the proteome, domain shuffling increases versatility, i.e. the number of distinct contexts in which a domain can occur. Here, we describe a comprehensive, genome-wide analysis of the relationship between these two processes. We observe a strong and robust correlation between domain versatility and abundance: domains that occur more often also have many different combination partners. This supports the view that domain recombination occurs in a random way. However, we do not observe all the different combinations that are expected from a simple random recombination scenario, and this is due to frequent duplication of specific domain combinations. When we simulate the evolution of the protein repertoire considering stochastic recombination of domains followed by extensive duplication of the combinations, we approximate the observed data well. Our analyses are consistent with a stochastic process that governs domain recombination and thus protein divergence with respect to domains within a polypeptide chain. At the same time, they support a scenario in which domain combinations are formed only once during the evolution of the protein repertoire, and are then duplicated to various extents. The extent of duplication of different combinations varies widely and, in nature, will depend on selection for the domain combination based on its function. Some of the pair-wise domain combinations that are highly duplicated also recur frequently with other partner domains, and thus represent evolutionary units larger than single protein domains, which we term "supra-domains".     

Microsatellite variation and recombination rate in the human genome

Background (purifying) selection on deleterious mutations is expected to remove linked neutral mutations from a population, resulting in a positive correlation between recombination rate and levels of neutral genetic variation, even for markers with high mutation rates. We tested this prediction of the background selection model by comparing recombination rate and levels of microsatellite polymorphism in humans. Published data for 28 unrelated Europeans were used to estimate microsatellite polymorphism (number of alleles, heterozygosity, and variance in allele size) for loci throughout the genome. Recombination rates were estimated from comparisons of genetic and physical maps. First, we analyzed 61 loci from chromosome 22, using the complete sequence of this chromosome to provide exact physical locations. These 61 microsatellites showed no correlation between levels of variation and recombination rate. We then used radiation-hybrid and cytogenetic maps to calculate recombination rates throughout the genome. Recombination rates varied by more than one order of magnitude, and most chromosomes showed significant suppression of recombination near the centromere. Genome-wide analyses provided no evidence for a strong positive correlation between recombination rate and polymorphism, although analyses of loci with at least 20 repeats suggested a weak positive correlation. Comparisons of microsatellites in lowest-recombination and highest-recombination regions also revealed no difference in levels of polymorphism. Together, these results indicate that background selection is not a major determinant of microsatellite variation in humans.     

The impact of recombination on nucleotide substitutions in the human genome

Unraveling the evolutionary forces responsible for variations of neutral substitution patterns among taxa or along genomes is a major issue for detecting selection within sequences. Mammalian genomes show large-scale regional variations of GC-content (the isochores), but the substitution processes at the origin of this structure are poorly understood. We analyzed the pattern of neutral substitutions in 1 Gb of primate non-coding regions. We show that the GC-content toward which sequences are evolving is strongly negatively correlated to the distance to telomeres and positively correlated to the rate of crossovers (R²=47%). This demonstrates that recombination has a major impact on substitution patterns in human, driving the evolution of GC-content. The evolution of GC-content correlates much more strongly with male than with female crossover rate, which rules out selectionist models for the evolution of isochores. This effect of recombination is most probably a consequence of the neutral process of biased gene conversion (BGC) occurring within recombination hotspots. We show that the predictions of this model fit very well with the observed substitution patterns in the human genome. This model notably explains the positive correlation between substitution rate and recombination rate. Theoretical calculations indicate that variations in population size or density in recombination hotspots can have a very strong impact on the evolution of base composition. Furthermore, recombination hotspots can create strong substitution hotspots. This molecular drive affects both coding and non-coding regions. We therefore conclude that along with mutation, selection and drift, BGC is one of the major factors driving genome evolution. Our results also shed light on variations in the rate of crossover relative to non-crossover events, along chromosomes and according to sex, and also on the conservation of hotspot density between human and chimp.     

Nonhomologous RNA-RNA recombination events at the 3' nontranslated region of the Sindbis virus genome: hot spots and utilization of nonviral sequences.

The mechanism of RNA-RNA recombination at the 3' nontranslated region (3'NTR) of the Sindbis virus (SIN) genome was studied by using nonreplicative RNA precursors. The 11.7-kb SIN genome was transcribed in vitro as two nonoverlapping RNA fragments. RNA-1 contained the entire 11.4-kb protein coding sequence of SIN and also carried an additional 1.8-kb nonviral sequence at its 3' end. RNA-2 carried the remaining 0.26 or 0.3 kb of the SIN genome containing the 3'NTR. Transfection of these two fragments into BHK cells resulted in vivo RNA-RNA recombination and release of infectious SIN recombinants. Eighteen plaque-purified recombinant viruses were sequenced to precisely map the RNA-RNA crossover sites at the 3'NTR. Sixteen of the 18 recombinants were found to be genetically heterogeneous at the 3'NTR. Two major clustered sites within the 3'NTR of RNA-2 were found to be fused to multiple locations on the nonviral sequence of RNA-1, resulting in insertions of 10 to 1,085 nucleotides at the 3'NTR. Sequence analysis of crossover sites suggested only limited homology and heteroduplex-forming capability between substrate RNAs. Analysis of additional 23 recombinant viruses generated by mutagenized donor and acceptor templates supports the occurrence of recombination hot spots on donor templates. Introduction of a 17-nucleotide rudimentary replicase recognition signal in the acceptor template alone did not induce the polymerase to reinitiate at the 17-nucleotide signal. Interestingly, deletion of a 24-nucleotide hot spot locus on the donor template abolished crossover events at one of the two sites and allowed the polymerase to reinitiate at the 17-nucleotide replicase recognition signal inserted at the acceptor template. The possible roles of RNA-protein and RNA-RNA interactions in the differential regulation of apparent pausing, template selection, and reinitiation are discussed.     

Differential activation of M26-containing meiotic recombination hot spots in Schizosaccharomyces pombe

Certain genomic loci, termed hot spots, are predisposed to undergo genetic recombination during meiosis at higher levels relative to the rest of the genome. The factors that specify hot-spot potential are not well understood. The M26 hot spot of Schizosaccharomyces pombe is dependent on certain trans activators and a specific nucleotide sequence, which can function as a hot spot in a position- and orientation-independent fashion within ade6. In this report we demonstrate that a linear element (LE) component, Rec10, has a function that is required for activation of some, but not all, M26-containing hot spots and from this we propose that, with respect to hot-spot activity, there are three classes of M26-containing sequences. We demonstrate that the localized sequence context in which the M26 heptamer is embedded is a major factor governing whether or not this Rec10 function is required for full hot-spot activation. Furthermore, we show that the rec10-144 mutant, which is defective in full activation of ade6-M26, but proficient for activation of other M26-containing hot spots, is also defective in the formation of LEs, suggesting an intimate link between higher-order chromatin structure and local influences on hot-spot activation.     

A high-resolution linkage map for the Z chromosome in chicken reveals hot spots for recombination

A comprehensive linkage map for chicken chromosome Z was constructed as the result of a large-scale screening of single nucleotide polymorphisms (SNPs). A total of 308 SNPs were assigned to Z based on the genotype distribution among 182 birds representing several populations. A linkage map comprising 210 markers and spanning 200.9 cM was established by analyzing a small Red junglefowl/White Leghorn intercross. There was excellent agreement between the linkage map for Z and a recently released assembly of the chicken genome (May 2006). Almost all SNPs assigned to chromosome Z in the present study are on Z in the new genome assembly. The remaining 12 loci are all found on unassigned contigs that can now be assigned to Z. The average recombination rate was estimated at 2.7 cM/Mb but there was a very uneven distribution of recombination events with both cold and hot spots of recombination. The existence of one of the major hot spots of recombination, located around position 39.4 Mb, was supported by the observed pattern of linkage disequilibrium. Thirteen markers from unassigned contigs were shown to be located on chromosome W. Three of these contigs included genes that have homologues on chromosome Z. The preliminary assignment of three more genes to the gene-poor W chromosome may be important for studies on the mechanism of sex determination and dosage compensation in birds.     

Analysis of biological features associated with meiotic recombination hot and cold spots in Saccharomyces cerevisiae

Meiotic recombination is not distributed uniformly throughout the genome. There are regions of high and low recombination rates called hot and cold spots, respectively. The recombination rate parallels the frequency of DNA double-strand breaks (DSBs) that initiate meiotic recombination. The aim is to identify biological features associated with DSB frequency. We constructed vectors representing various chromatin and sequence-based features for 1179 DSB hot spots and 1028 DSB cold spots. Using a feature selection approach, we have identified five features that distinguish hot from cold spots in Saccharomyces cerevisiae with high accuracy, namely the histone marks H3K4me3, H3K14ac, H3K36me3, and H3K79me3; and GC content. Previous studies have associated H3K4me3, H3K36me3, and GC content with areas of mitotic recombination. H3K14ac and H3K79me3 are novel predictions and thus represent good candidates for further experimental study. We also show nucleosome occupancy maps produced using next generation sequencing exhibit a bias at DSB hot spots and this bias is strong enough to obscure biologically relevant information. A computational approach using feature selection can productively be used to identify promising biological associations. H3K14ac and H3K79me3 are novel predictions of chromatin marks associated with meiotic DSBs. Next generation sequencing can exhibit a bias that is strong enough to lead to incorrect conclusions. Care must be taken when interpreting high throughput sequencing data where systematic biases have been documented.     

人类基因组中突变对紧邻碱基的依赖性与重组率

减数分裂重组通过基因转变、碱基替换等方式影响基因组进化。紧邻碱基对突变偏好性有很强的影响,但该“紧邻碱基效应”如何随重组率变化有待深入研究。本文提出基于条件互信息(Conditional mutual information)量化突变对紧邻碱基依赖性的方法,并利用人类SNP等相关数据,分析重组率如何影响突变对紧邻碱基的依赖性。结果表明:在全基因组水平上, SNP位点上的突变对紧邻碱基的依赖性(即平均条件互信息)随着重组率的增加而增加;具体而言,当SNPs两侧碱基为A/G、C/G或C/T时,随着重组率的增加突变偏向性增强,但两侧碱基为A/A或T/T时,重组率对SNP突变偏向性产生抑制作用;另外,重组率越高,外显子与基因间区SNP的突变偏好性越强;而内含子区域SNP的突变偏好受到高重组率的抑制。结果有助于深入理解减数分裂重组如何影响基因组进化。