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.     

Recombinational and mutational hotspots within the human lipoprotein lipase gene

Here an analysis is presented of the roles of recombination and mutation in shaping previously determined haplotype variation in 9.7 kb of genomic DNA sequence from the human lipoprotein lipase gene (LPL), scored in 71 individuals from three populations: 24 African Americans, 24 Finns, and 23 non-Hispanic whites. Recombination and gene-conversion events inferred from data on 88 haplotypes that were defined by 69 variable sites were tested. The analysis revealed 29 statistically significant recombination events and one gene-conversion event. The recombination events were concentrated in a 1.9-kb region, near the middle of the segment, that contains a microsatellite and a pair of tandem and complementary mononucleotide runs; both the microsatellite and the runs show length variation. An analysis of site variation revealed that 9.6% of the nucleotides at CpG sites were variable, as were 3% of the nucleotides found in mononucleotide runs of >/=5 nucleotides, 3% of the nucleotides found 相似文献   

Human Triallelic Sites: Evidence for a New Mutational Mechanism?

Most SNPs in the human genome are biallelic; however, there are some sites that are triallelic. We show here that there are approximately twice as many triallelic sites as we would expect by chance. This excess does not appear to be caused by natural selection or mutational hotspots. Instead we propose that a new mutation can induce another mutation either within the same individual or subsequently during recombination. We provide evidence for this model by showing that the rarer two alleles at triallelic sites tend to cluster on phylogenetic trees of human haplotypes. However, we find no association between the density of triallelic sites and the rate of recombination, which leads us to suggest that triallelic sites might be generated by the simultaneous production of two new mutations within the same individual on the same genetic background. Under this model we estimate that simultaneous mutation contributes ∼3% of all distinct SNPs. We also show that there is a twofold excess of adjacent SNPs. Approximately half of these seem to be generated simultaneously since they have identical minor allele frequencies. We estimate that the mutation of adjacent nucleotides accounts for a little less than 1% of all SNPs.ALTHOUGH the density of biallelic SNPs in the human genome is reasonably low, there are some sites that have three (triallelic sites) or even four nucleotides segregating in the human population. We show here that there are approximately twice as many triallelic sites as we would expect by chance. There are at least three mutational mechanisms that could potentially generate such an excess of triallelic sites. First, some sites may be hypermutable, and if the mutation rate of at least two pathways (e.g., C → T and C → A) is elevated at such sites, then there will be an excess of triallelic sites. The mutation rate of a site is known to depend upon the adjacent nucleotides, the best known example being the CpG dinucleotide (Coulondre et al. 1978; Bird 1980) at which the frequency of both transition and transversion mutations is elevated. However, other adjacent nucleotides also influence the mutation rate (Blake et al. 1992; Zhao et al. 2003; Hwang and Green 2004). Furthermore, we have recently shown that there is variation in the mutation rate that does not depend upon the identity of the adjacent nucleotides or any specific context (Hodgkinson et al. 2009).Second, it is possible that two of the alleles at a triallelic site are generated simultaneously within a single individual. Point mutations are generally assumed to involve the production of a single new allele per mutation event at a rate that is governed by the effects mentioned above. However, it is not difficult to imagine mechanisms that might induce mutations on both strands of the DNA duplex; for example, the presence of a base mismatch may itself be unstable, so we might go from a G-C base pair to a G-A, which then may mutate to C-A; if DNA replication reads through this mismatch, the G allele will have mutated to both C and T. Alternatively, the mutation may occur across both strands of the duplex at the same time, possibly as a result of a chemical or radiation event. Third, in a similar manner, we might imagine a single SNP inducing subsequent mutations if base mismatches are formed during recombination in heteroduplex DNA.Here we attempt to identify the cause of the excess of triallelic sites by analyzing sequence data around triallelic sites.     

Trans-Regulation of Mouse Meiotic Recombination Hotspots by Rcr1

Meiotic recombination is required for the orderly segregation of chromosomes during meiosis and for providing genetic diversity among offspring. Among mammals, as well as yeast and higher plants, recombination preferentially occurs at highly delimited chromosomal sites 1–2 kb long known as hotspots. Although considerable progress has been made in understanding the roles various proteins play in carrying out the molecular events of the recombination process, relatively little is understood about the factors controlling the location and relative activity of mammalian recombination hotspots. To search for trans-acting factors controlling the positioning of recombination events, we compared the locations of crossovers arising in an 8-Mb segment of a 100-Mb region of mouse Chromosome 1 (Chr 1) when the longer region was heterozygous C57BL/6J (B6) × CAST/EiJ (CAST) and the remainder of the genome was either similarly heterozygous or entirely homozygous B6. The lack of CAST alleles in the remainder of the genome resulted in profound changes in hotspot activity in both females and males. Recombination activity was lost at several hotspots; new, previously undetected hotspots appeared; and still other hotspots remained unaffected, indicating the presence of distant trans-acting gene(s) whose CAST allele(s) activate or suppress the activity of specific hotspots. Testing the activity of three activated hotspots in sperm samples from individual male progeny of two genetic crosses, we identified a single trans-acting regulator of hotspot activity, designated Rcr1, that is located in a 5.30-Mb interval (11.74–17.04 Mb) on Chr 17. Using an Escherichia coli cloning assay to characterize the molecular products of recombination at two of these hotspots, we found that Rcr1 controls the appearance of both crossover and noncrossover gene conversion events, indicating that it likely controls the sites of the double-strand DNA breaks that initiate the recombination process.     

A hotspot of spontaneous and UV-induced illegitimate recombination during formation ofλbio transducing phage

To study the mechanism of spontaneous and UV-induced illegitimate recombination, we examined the formation of thebio specialized transducing phage inEscherichia coli. Because mostbio transducing phages have double defects in thered andgam genes and have the capacity to form a plaque on anE. coli P2 lysogen (Spi^– phenotype), we selectedbio transducing phage by their Spi^– phenotype, rather than using thebio marker. We determined sequences of recombination junctions ofbio transducing phages isolated with or without UV irradiation and deduced sequences of parental recombination sites. The recombination sites were widely distributed onE. coli bio and DNAs, except for a hotspot which accounts for 57% of UV-inducedbio transducing phages and 77% of spontaneously inducedbio transducing phages. The hotspot sites onE. coli and DNAs shared a short homology of 9 bp. In addition, we detected direct repeat sequences of 8 by within and near both thebio and hotspots. ArecA mutation did not affect the frequency of the recombination at the hotspot, indicating that this recombination is not a variant ofrecA-dependent homologous recombination. We discuss a model in which the short homology as well as the direct repeats play essential roles in illegitimate recombination at the hotspot.     

Inference of the Properties of the Recombination Process from Whole Bacterial Genomes

Patterns of linkage disequilibrium, homoplasy, and incompatibility are difficult to interpret because they depend on several factors, including the recombination process and the population structure. Here we introduce a novel model-based framework to infer recombination properties from such summary statistics in bacterial genomes. The underlying model is sequentially Markovian so that data can be simulated very efficiently, and we use approximate Bayesian computation techniques to infer parameters. As this does not require us to calculate the likelihood function, the model can be easily extended to investigate less probed aspects of recombination. In particular, we extend our model to account for the bias in the recombination process whereby closely related bacteria recombine more often with one another. We show that this model provides a good fit to a data set of Bacillus cereus genomes and estimate several recombination properties, including the rate of bias in recombination. All the methods described in this article are implemented in a software package that is freely available for download at http://code.google.com/p/clonalorigin/.     

Bayesian Inference of Shared Recombination Hotspots Between Humans and Chimpanzees

Recombination generates variation and facilitates evolution. Recombination (or lack thereof) also contributes to human genetic disease. Methods for mapping genes influencing complex genetic diseases via association rely on linkage disequilibrium (LD) in human populations, which is influenced by rates of recombination across the genome. Comparative population genomic analyses of recombination using related primate species can identify factors influencing rates of recombination in humans. Such studies can indicate how variable hotspots for recombination may be both among individuals (or populations) and over evolutionary timescales. Previous studies have suggested that locations of recombination hotspots are not conserved between humans and chimpanzees. We made use of the data sets from recent resequencing projects and applied a Bayesian method for identifying hotspots and estimating recombination rates. We also reanalyzed SNP data sets for regions with known hotspots in humans using samples from the human and chimpanzee. The Bayes factors (BF) of shared recombination hotspots between human and chimpanzee across regions were obtained. Based on the analysis of the aligned regions of human chromosome 21, locations where the two species show evidence of shared recombination hotspots (with high BFs) were identified. Interestingly, previous comparative studies of human and chimpanzee that focused on the known human recombination hotspots within the β-globin and HLA regions did not find overlapping of hotspots. Our results show high BFs of shared hotspots at locations within both regions, and the estimated locations of shared hotspots overlap with the locations of human recombination hotspots obtained from sperm-typing studies.     

Population bottlenecks in Polynesia revealed by minisatellites

Summary Tandem-repetitive highly variable loci in the human genome (minisatellites) have been used in gene mapping and as DNA fingerprints, but they have not yet found much application in population genetics. We have investigated the capacity of six minisatellites to discriminate between four populations in Oceania. We find that in comparison to Melanesians, Polynesians have a significant loss of heterozygosity (or gene diversity), not noted using more traditional markers. We show also that the number of alleles, the allele distribution and the mutation rates at the Polynesian minisatellite loci do not deviate from those predicted by the neutral mutation/infinite allele model. The low gene diversity is therefore likely to be a result of the maintenance of small population sizes and bottleneck effects during the colonization of the Pacific.     

Molecular Evolution of the Hepatitis Delta Virus Antigen Gene: Recombination or Positive Selection?

We present the statistical analysis of diversifying selective pressures on the hepatitis D antigen gene (HDAg). Thirty-three distinct HDAg sequences from subtypes I, II, and III were tested for positive selection using maximum likelihood methods based on models of codon substitution that allow variable selective pressures across sites. Such methods have been shown to be sufficiently accurate and successful in detecting positive selection in a variety of viral and nonviral protein-coding genes. About 11% of codon sites in HDAg were estimated to be under diversifying selection. Remarkably, most of the residues predicted to evolve under positive selection were located in the immunogenic domain and the N-terminus region with reported antigenic activity. These sites are potential targets of the hosts immune response. Identification of residues mutating to escape immune recognition may help to distinguish the most virulent strains and aid vaccine design. Possible interplay between positive selection and recombination on the gene is discussed but no significant evidence for recombination was found.This article contains online supplementary material.Reviewing Editor: Dr. Nicolas Galtier     

The two-locus ancestral graph in a subdivided population: convergence as the number of demes grows in the island model

We study the ancestral recombination graph for a pair of sites in a geographically structured population. In particular, we consider the limiting behavior of the graph, under Wrights island model, as the number of subpopulations, or demes, goes to infinity. After an instantaneous sample-size adjustment, the graph becomes identical to the two-locus graph in an unstructured population, but with a time scale that depends on the migration rate and the deme size. Interestingly, when migration is gametic, this rescaling of time increases the population mutation rate but does not affect the population recombination rate. We compare this to the case of a partially-selfing population, in which both mutation and recombination depend on the selfing rate. Our result for gametic migration holds both for finite-sized demes, and in the limit as the deme size goes to infinity. However, when migration occurs during the diploid phase of the life cycle and demes are finite in size, the population recombination rate does depend on the migration rate, in a way that is reminiscent of partial selfing. Simulations imply that convergence to a rescaled panmictic ancestral recombination graph occurs for any number of sites as the number of demes approaches infinity.Send offprint request to: Sabin LessardS. Lessard was supported by grants from the Natural Sciences and Research Council of Canada, the Fonds Québécois de la Recherche sur la Nature et les Technologies, and the Université de Montréal.J. Wakeley was supported by a Career Award (DEB-0133760) and by a grant (DEB-9815367) from the National Science Foundation.     

Mutational hotspots in the TP53 gene and, possibly, other tumor suppressors evolve by positive selection

Background  

The mutation spectra of the TP53 gene and other tumor suppressors contain multiple hotspots, i.e., sites of non-random, frequent mutation in tumors and/or the germline. The origin of the hotspots remains unclear, the general view being that they represent highly mutable nucleotide contexts which likely reflect effects of different endogenous and exogenous factors shaping the mutation process in specific tissues. The origin of hotspots is of major importance because it has been suggested that mutable contexts could be used to infer mechanisms of mutagenesis contributing to tumorigenesis.     

Suppression of gamma ray-induced illegitimate recombination in Escherichia coli by the DNA-binding protein H-NS

To study the mechanism of gamma-ray-induced illegitimate recombination, we examined the formation of lambdabio transducing phage in Escherichia coli after gamma-ray irradiation. We show that gamma-ray irradiation enhances the formation of lambdabio transducing phage during prophage induction. Moreover, an hns mutation synergistically enhanced the incidence of lambda-ray-induced illegitimate recombination. Next we determined the sequences at the recombination junctions of the lambdabio transducing phages induced by gamma-ray irradiation. Most of the recombination sites coincided with known hotspots. Among them, hotspot I accounted for 67% and 77% of gamma-ray-induced lambdabio transducing phages in the wild type and the hns mutant, respectively. Therefore, the recombination sites appear to occur mostly at hotspot I or at other hotspots, but rarely at non-hotspot sites. These results suggest that types of DNA damage other than the double-strand breaks induced at random sites are mainly responsible for the introduction of the site-specific or region-specific DNA double strand breaks that lead to recombination at the hotspots. The results also showed that the recombination events took place between DNA sequences possessing short stretches of homology. H-NS protein, which binds to curved DNA, suppresses illegitimate recombination in the presence and absence of gamma-ray irradiation. Models for gamma-ray-induced illegitimate recombination are discussed.     

A New Isolation with Migration Model along Complete Genomes Infers Very Different Divergence Processes among Closely Related Great Ape Species

We present a hidden Markov model (HMM) for inferring gradual isolation between two populations during speciation, modelled as a time interval with restricted gene flow. The HMM describes the history of adjacent nucleotides in two genomic sequences, such that the nucleotides can be separated by recombination, can migrate between populations, or can coalesce at variable time points, all dependent on the parameters of the model, which are the effective population sizes, splitting times, recombination rate, and migration rate. We show by extensive simulations that the HMM can accurately infer all parameters except the recombination rate, which is biased downwards. Inference is robust to variation in the mutation rate and the recombination rate over the sequence and also robust to unknown phase of genomes unless they are very closely related. We provide a test for whether divergence is gradual or instantaneous, and we apply the model to three key divergence processes in great apes: (a) the bonobo and common chimpanzee, (b) the eastern and western gorilla, and (c) the Sumatran and Bornean orang-utan. We find that the bonobo and chimpanzee appear to have undergone a clear split, whereas the divergence processes of the gorilla and orang-utan species occurred over several hundred thousands years with gene flow stopping quite recently. We also apply the model to the Homo/Pan speciation event and find that the most likely scenario involves an extended period of gene flow during speciation.     

Redirecting meiotic DNA break hotspot determinant proteins alters localized spatial control of DNA break formation and repair

During meiosis, DNA double-strand breaks (DSBs) are formed at high frequency at special chromosomal sites, called DSB hotspots, to generate crossovers that aid proper chromosome segregation. Multiple chromosomal features affect hotspot formation. In the fission yeast S. pombe the linear element proteins Rec25, Rec27 and Mug20 are hotspot determinants – they bind hotspots with high specificity and are necessary for nearly all DSBs at hotspots. To assess whether they are also sufficient for hotspot determination, we localized each linear element protein to a novel chromosomal site (ade6 with lacO substitutions) by fusion to the Escherichia coli LacI repressor. The Mug20-LacI plus lacO combination, but not the two separate lac elements, produced a strong ade6 DSB hotspot, comparable to strong endogenous DSB hotspots. This hotspot had unexpectedly low ade6 recombinant frequency and negligible DSB hotspot competition, although like endogenous hotspots it manifested DSB interference. We infer that linear element proteins must be properly placed by endogenous functions to impose hotspot competition and proper partner choice for DSB repair. Our results support and expand our previously proposed DSB hotspot-clustering model for local control of meiotic recombination.     

Comparative analysis of complete chloroplast genome sequences of two subtropical trees, <Emphasis Type="Italic">Phoebe sheareri</Emphasis> and <Emphasis Type="Italic">Phoebe omeiensis</Emphasis> (Lauraceae)

Phoebe is an economically important genus from the family Lauraceae. It is widely distributed in tropical and subtropical Asia, but systematics of the genus is unclear, and currently there is no species-level phylogeny. Here, we determined the complete chloroplast genome sequences of two species with long-range PCR and next genome sequencing technologies, and identified mutation sites and highly variable regions. These highly variable sites were used to reconstruct the phylogeny. The plastomes of Phoebe sheareri and P. omeiensis were 152, 876, and 152, 855 bp, respectively. Comparative genomic analysis indicated that there are 222 mutation sites including 146 substitutions, 73 indels, and 3 microinversions in both plastomes. Fifty-six single-nucleotide changes were identified in gene-coding regions, and 45 microsatellite sites were found for use in species identification. Fourteen divergence hotspots of 38 variable regions were located. Phylogeny was reconstructed using a Bayesian and maximum likelihood approach for 12 Phoebe species and other five related Lauraceae based on 15 of the highly variable regions including accD-psaI, atpB-rbcL, ndhC-trnV, ndhF-rpl32, petA-psbJ, psaA, psbA-trnH, rbcL, rps8-rpl14, rps16-trnQ, rpl32-trnL, trnC-petN, trnL-trnF, trnS-trnG, and ycf1 indicated that variability in the chloroplast regions proposed as variable is enough to detect divergence events among 12 taxa of Phoebe, and that maybe also useful to help to elucidate further relationships among other taxa of the genus.     

DNA sequences required to induce localized conversion in Streptococcus pneumoniae transformation

Summary In pneumococcal transformation a particular point mutation belonging to the amiA locus is able markedly to enhance recombination frequency when crossed with any other markers of this gene. This results from a polarized conversion of the mutation towards the wild-type sequence. In this report, by site-directed oligonucleotide mutagenesis, we have generated a series of mutants showing various degrees of conversion. We have found that the substitution 5-ATTCAT5-ATTAAT is a sufficient signal for localized conversion. Changing individual bases within this sequence results in decreased conversion frequencies to levels that depend on the mutation, suggesting that there is a family to related sequences which may act as a substrate for a conversion system. Moreover, the length over which this conversion occurs has been estimated to be 12 base pairs on the average.     

Bayesian Estimation of Positively Selected Sites

In protein-coding DNA sequences, historical patterns of selection can be inferred from amino acid substitution patterns. High relative rates of nonsynonymous to synonymous changes (=d _N /d _S ) are a clear indicator of positive, or directional, selection, and several recently developed methods attempt to distinguish these sites from those under neutral or purifying selection. One method uses an empirical Bayesian framework that accounts for varying selective pressures across sites while conditioning on the parameters of the model of DNA evolution and on the phylogenetic history. We describe a method that identifies sites under diversifying selection using a fully Bayesian framework. Similar to earlier work, the method presented here allows the rate of nonsynonymous to synonymous changes to vary among sites. The significant difference in using a fully Bayesian approach lies in our ability to account for uncertainty in parameters including the tree topology, branch lengths, and the codon model of DNA substitution. We demonstrate the utility of the fully Bayesian approach by applying our method to a data set of the vertebrate -globin gene. Compared to a previous analysis of this data set, the hierarchical model found most of the same sites to be in the positive selection class, but with a few striking exceptions.     

SSV1-encoded site-specific recombination system in Sulfolobus shibatae

Summary We present evidence for the existence of a conservative site-specific recombination system in Archaea by demonstrating integrative recombination of Sulfolobus shibatae virus SSV1 DNA with the host chromosome, catalysed by the SSVI-encoded integrase in vitro. The putative int gene of SSV1 was expressed in Escherichia coli yielding a protein of about 39 kDa. This protein alone efficiently recombined linear DNA substrates containing chromosomal (attA) and viral (attP) attachment sites; recombination with either negatively or positively supercoiled SSV1 DNA was less efficient. Intermolecular attA × attA and attP × attP recombination was also promoted by the SSV integrase. The invariant 44 by common attachment core present in all att sites contained sufficient information to allow recombination, whilst the flanking sequences effected the efficiency. These features clearly distinguish the SSV1 — encoded site — specific recombination system from others and make it suitable for the study of regulatory mechanisms of SSV1 genome — host chromosome interaction and investigations of the evolution of the recombination machinery.     

Active and Inactive Transplacement of the M26 Recombination Hotspot in Schizosaccharomyces Pombe

The ade6-M26 mutation of the fission yeast Schizosaccharomyces pombe creates a meiotic recombination hotspot that elevates ade6 intragenic recombination ~10-15-fold. A heptanucleotide sequence including the M26 point mutation is required but not sufficient for hotspot activity. We studied the effects of plasmid and chromosomal context on M26 hotspot activity. The M26 hotspot was inactive on a multicopy plasmid containing M26 embedded within 3.0 or 5.9 kb of ade6 DNA. Random S. pombe genomic fragments totaling ~7 Mb did not activate the M26 hotspot on a plasmid. M26 hotspot activity was maintained when 3.0-, 4.4-, and 5.9-kb ade6-M26 DNA fragments, with various amounts of non-S. pombe plasmid DNA, were integrated at the ura4 chromosomal locus, but only in certain configurations relative to the ura4 gene and the cointegrated plasmid DNA. Several integrations created new M26-independent recombination hotspots. In all cases the non-ade6 DNA was located >1 kb from the M26 site, and in some cases >2 kb. Because the chromosomal context effect was transmitted over large distances, and did not appear to be mediated by a single discrete DNA sequence element, we infer that the local chromatin structure has a pronounced effect on M26 hotspot activity.