Mechanistic View and Genetic Control of DNA Recombination during Meiosis.

Meiotic recombination is essential for fertility and allelic shuffling. Canonical recombination models fail to capture the observed complexity of meiotic recombinants. Here, by combining genome-wide meiotic heteroduplex DNA patterns with meiotic DNA double-strand break (DSB) sites, we show that part of this complexity results from frequent template switching during synthesis-dependent strand annealing that yields noncrossovers and from branch migration of double Holliday junction (dHJ)-containing intermediates that mainly yield crossovers. This complexity also results from asymmetric positioning of crossover intermediates relative to the initiating DSB and Msh2-independent conversions promoted by the suspected dHJ resolvase Mlh1-3 as well as Exo1 and Sgs1. Finally, we show that dHJ resolution is biased toward cleavage of the pair of strands containing newly synthesized DNA near the junctions and that this bias can be decoupled from the crossover-biased dHJ resolution. These properties are likely conserved in eukaryotes containing ZMM proteins, which includes mammals.

Comparative analysis of the sensitivity of metagenomic sequencing and PCR to detect a biowarfare simulant (Bacillus atrophaeus) in soil samples.

To evaluate the sensitivity of high-throughput DNA sequencing for monitoring biowarfare agents in the environment, we analysed soil samples inoculated with different amounts of Bacillus atrophaeus, a surrogate organism for Bacillus anthracis. The soil samples considered were a poorly carbonated soil of the silty sand class, and a highly carbonated soil of the silt class. Control soil samples and soil samples inoculated with 10, 103, or 105 cfu were processed for DNA extraction. About 1% of the DNA extracts was analysed through the sequencing of more than 108 reads. Similar amounts of extracts were also studied for Bacillus atrophaeus DNA content by real-time PCR. We demonstrate that, for both soils, high-throughput sequencing is at least equally sensitive than real-time PCR to detect Bacillus atrophaeus DNA. We conclude that metagenomics allows the detection of less than 10 ppm of DNA from a biowarfare simulant in complex environmental samples.

Genome data on the extinct Bison schoetensacki establish it as a sister species of the extant European bison (Bison bonasus).

BACKGROUND: The European bison (Bison bonasus), now found in Europe and the Caucasus, has been proposed to originate either from the extinct steppe/extant American bison lineage or from the extinct Bison schoetensacki lineage. Bison schoetensacki remains are documented in Eurasian Middle Pleistocene sites, but their presence in Upper Pleistocene sites has been questioned. Despite extensive genetic studies carried out on the steppe and European bison, no remains from the fossil record morphologically identified as Bison schoetensacki has been analyzed up to now. RESULTS: In this paper, we analyzed a 36,000-year-old Bison schoetensaki bone sample from the Siréjol cave (France) and a cave hyena coprolite (fossilized feces) found in a nearby cave and containing large amounts of Bovinae DNA. We show that the Bovinae mitochondrial DNA sequences from both samples, including a complete mitochondrial genome sequence, belong to a clade recently reported in the literature. This clade only includes ancient bison specimens without taxonomic identification and displays a sister relationship with the extant European bison. The genetic proximity of Bison schoetensacki with specimens from this clade is corroborated by the analysis of nuclear DNA single nucleotide polymorphisms. CONCLUSIONS: This work provides genetic evidence supporting the continuing presence of Bison schoetensacki up to the Upper Pleistocene. Bison schoetensacki turns out to be a sister species of Bison bonasus, excluding the steppe bison Bison priscus as a direct ancestor of the European bison.

Concerted action of the MutLß heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion.

Gene conversions resulting from meiotic recombination are critical in shaping genome diversification and evolution. How the extent of gene conversions is regulated is unknown. Here we show that the budding yeast mismatch repair related MutLß complex, Mlh1-Mlh2, specifically interacts with the conserved meiotic Mer3 helicase, which recruits it to recombination hotspots, independently of mismatch recognition. This recruitment is essential to limit gene conversion tract lengths genome-wide, without affecting crossover formation. Contrary to expectations, Mer3 helicase activity, proposed to extend the displacement loop (D-loop) recombination intermediate, does not influence the length of gene conversion events, revealing non-catalytical roles of Mer3. In addition, both purified Mer3 and MutLß preferentially recognize D-loops, providing a mechanism for limiting gene conversion in vivo. These findings show that MutLß is an integral part of a new regulatory step of meiotic recombination, which has implications to prevent rapid allele fixation and hotspot erosion in populations.

Hunting the Extinct Steppe Bison (Bison priscus) Mitochondrial Genome in the Trois-Frères Paleolithic Painted Cave.

Despite the abundance of fossil remains for the extinct steppe bison (Bison priscus), an animal that was painted and engraved in numerous European Paleolithic caves, a complete mitochondrial genome sequence has never been obtained for this species. In the present study we collected bone samples from a sector of the Trois-Frères Paleolithic cave (Ariège, France) that formerly functioned as a pitfall and was sealed before the end of the Pleistocene. Screening the DNA content of the samples collected from the ground surface revealed their contamination by Bos DNA. However, a 19,000-year-old rib collected on a rock apart the pathway delineated for modern visitors was devoid of such contaminants and reproducibly yielded Bison priscus DNA. High-throughput shotgun sequencing combined with conventional PCR analysis of the rib DNA extract enabled to reconstruct a complete mitochondrial genome sequence of 16,318 bp for the extinct steppe bison with a 10.4-fold coverage. Phylogenetic analyses robustly established the position of the Bison priscus mitochondrial genome as basal to the clade delineated by the genomes of the modern American Bison bison. The extinct steppe bison sequence, which exhibits 93 specific polymorphisms as compared to the published Bison bison mitochondrial genomes, provides an additional resource for the study of Bovinae specimens. Moreover this study of ancient DNA delineates a new research pathway for the analysis of the Magdalenian Trois-Frères cave.

Models for the evolution of GC content in asexual fungi Candida albicans and C. dubliniensis.

Although guanine-cytosine (GC)-biased gene conversion (gBGC) following meiotic recombination seems the most probable mechanism accounting for large-scale variations in GC content for many eukaryotes, it cannot explain such variations for organisms belonging to ancient asexual lineages, such as the pathogenic fungi Candida albicans and C. dubliniensis. Analysis of the substitution patterns for these two species reveals a strong anticorrelation between the synonymous transition rates at third codon positions. I propose two models that can account for this observation. According to the first model, the evolution of GC content is driven by gBGC linked to mitotic recombination, either associated with parasexuality or with damage repair. Variations in the GC content thus reflect variations in the strength of gBGC, presumably variations in the mitotic recombination rate. According to the second model, the evolution of GC content is driven by misincorporation errors during the process of DNA replication in S phase. This model proposes that variations in GC content are due to variations in the proportions of dCTPs and dGTPs at the time when sequences are replicated. Experimental data regarding mitotic recombination rates or the variations of dCTPs and dGTPs during S phase are required to validate definitively one of the two models, but in any case, the fit of the models to the data suggests that C. albicans and C. dubliniensis constitute so far unique examples of GC content evolution driven either by mitotic recombination or replicative errors.

Asymmetry indices for analysis and prediction of replication origins in eukaryotic genomes.

DNA replication was recently shown to induce the formation of compositional skews in the genomes of the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis. In this work, I have characterized further GC and TA skew variations in the vicinity of S. cerevisiae replication origins and termination sites, and defined asymmetry indices for origin analysis and prediction. The presence of skew jumps at some termination sites in the S. cerevisiae genome was established. The majority of S. cerevisiae replication origins are marked by an oriented consensus sequence called ACS, but no evidence could be found for asymmetric origin firing that would be linked to ACS orientation. Asymmetry indices related to GC and TA skews were defined, and a global asymmetry index I(GC,TA) was described. I(GC,TA) was found to strongly correlate with origin efficiency in S. cerevisiae and to allow the determination of sets of intergenes significantly enriched in origin loci. The generalized use of asymmetry indices for origin prediction in naive genomes implies the determination of the direction of the skews, i.e. the identification of which strand, leading or lagging, is enriched in G and which one is enriched in T. Recent work indicates that in Candida albicans and in several related species, centromeres contain early and efficient replication origins. It has been proposed that the skew jumps observed at these positions would reflect the activity of these origins, thus allowing to determine the direction of the skews in these genomes. However, I show here that the skew jumps at C. albicans centromeres are not related to replication and that replication-associated GC and TA skews in C. albicans have in fact the opposite directions of what was proposed.

DNA replication induces compositional biases in yeast.

Asymmetries intrinsic to the process of DNA replication are expected to cause differences in the substitution patterns of the leading and the lagging strands and to induce compositional biases. These biases have been detected in the majority of eubacterial genomes but rarely in eukaryotes. Only in the human genome, the activity of a minority of replication origins seems to generate compositional biases. In this work, we provide evidence for replication-associated GC and TA skews in the genomes of two yeast species, Saccharomyces cerevisiae and Kluyveromyces lactis, whereas the data for the Schizosaccharomyces pombe genome are less conclusive. In contrast with the genomes of Homo sapiens and of the majority of eubacteria, the leading strand is enriched in cytosine and adenine in both S. cerevisiae and K. lactis. We observed significant variations across the interorigin intervals of several substitution rates in the S. cerevisiae lineage since its divergence from S. paradoxus. We also found that the S. cerevisiae genome is far from compositional equilibrium and that its present compositional biases are due to substitution rates operating before its divergence from S. paradoxus. Finally, we observed that replication and transcription tend to be cooriented in the S. cerevisiae genome, especially for genes encoding subunits of protein complexes. Taken together, our results suggest that replication-related compositional biases may be a feature of many eukaryotic genomes despite the stochastic nature of the firing of replication origins in these genomes.

Giant yeast cells with nonrecyclable ribonucleotide reductase.

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides and thereby provides the precursors required for DNA synthesis and repair. In an attempt to test cell resistance to a permanent replicational stress, we constructed a mutant Saccharomyces cerevisiae strain containing exclusively nonrecyclable catalytic subunits of RNR that become inactivated following the reduction of one ribonucleoside diphosphate. In this rnr1C883A rnr3Δ mutant, the synthesis of each deoxyribonucleotide thus requires the production of one Rnr1C883A protein, which means that 26 million Rnr1C883A proteins (half the protein complement of a wild-type cell) have to be produced during each cell cycle. rnr1C883A rnr3Δ cells grow under constant replicational stress, as evidenced by the constitutive activation of the checkpoint effector Rad53, and their S phase is considerably extended compared to the wild type. rnr1C883A rnr3Δ mutants also display additional abnormalities such as a median cell volume increased by a factor of 8, and the presence of massive inclusion bodies. However, they exhibit a good plating efficiency and can be propagated indefinitely. rnr1C883A rnr3Δ cells, which can be used as a protein overexpression system, thus illustrate the robustness of S. cerevisiae to multiple physiological parameters.

A simple model for the influence of meiotic conversion tracts on GC content.

A strong correlation between GC content and recombination rate is observed in many eukaryotes, which is thought to be due to conversion events linked to the repair of meiotic double-strand breaks. In several organisms, the length of conversion tracts has been shown to decrease exponentially with increasing distance from the sites of meiotic double-strand breaks. I show here that this behavior leads to a simple analytical model for the evolution and the equilibrium state of the GC content of sequences devoid of meiotic double-strand break sites. In the yeast Saccharomyces cerevisiae, meiotic double-strand breaks are practically excluded from protein-coding sequences. A good fit was observed between the predictions of the model and the variations of the average GC content of the third codon position (GC3) of S. cerevisiae genes. Moreover, recombination parameters that can be extracted by fitting the data to the model coincide with experimentally determined values. These results thus indicate that meiotic recombination plays an important part in determining the fluctuations of GC content in yeast coding sequences. The model also accounted for the different patterns of GC variations observed in the genes of Candida species that exhibit a variety of sexual lifestyles, and hence a wide range of meiotic recombination rates. Finally, the variations of the average GC3 content of human and chicken coding sequences could also be fitted by the model. These results suggest the existence of a widespread pattern of GC variation in eukaryotic genes due to meiotic recombination, which would imply the generality of two features of meiotic recombination: its association with GC-biased gene conversion and the quasi-exclusion of meiotic double-strand breaks from coding sequences. Moreover, the model points out to specific constraints on protein fragments encoded by exon terminal sequences, which are the most affected by the GC bias.

A strategy for interaction site prediction between phospho-binding modules and their partners identified from proteomic data.

Small and large scale proteomic technologies are providing a wealth of potential interactions between proteins bearing phospho-recognition modules and their substrates. Resulting interaction maps reveal such a dense network of interactions that the functional dissection and understanding of these networks often require to break specific interactions while keeping the rest intact. Here, we developed a computational strategy, called STRIP, to predict the precise interaction site involved in an interaction with a phospho-recognition module. The method was validated by a two-hybrid screen carried out using the ForkHead Associated (FHA)1 domain of Rad53, a key protein of Saccharomyces cerevisiae DNA checkpoint, as a bait. In this screen we detected 11 partners, including Cdc7 and Cdc45, essential components of the DNA replication machinery. FHA domains are phospho-threonine binding modules and the threonines involved in both interactions could be predicted using the STRIP strategy. The threonines T484 and T189 in Cdc7 and Cdc45, respectively, were mutated and loss of binding could be monitored experimentally with the full-length proteins. The method was further tested for the analysis of 63 known Rad53 binding partners and provided several key insights regarding the threonines likely involved in these interactions. The STRIP method relies on a combination of conservation, phosphorylation likelihood, and binding specificity criteria and can be accessed via a web interface at http://biodev.extra.cea.fr/strip/.

Universal temporal profile of replication origin activation in eukaryotes.

Although replication proteins are conserved among eukaryotes, the sequence requirements for replication initiation differ between species. In all species, however, replication origins fire asynchronously throughout S phase. The temporal program of origin firing is reproducible in cell populations but largely probabilistic at the single-cell level. The mechanisms and the significance of this program are unclear. Replication timing has been correlated with gene activity in metazoans but not in yeast. One potential role for a temporal regulation of origin firing is to minimize fluctuations in replication end time and avoid persistence of unreplicated DNA in mitosis. Here, we have extracted the population-averaged temporal profiles of replication initiation rates for S. cerevisiae, S. pombe, D. melanogaster, X. laevis and H. sapiens from genome-wide replication timing and DNA combing data. All the profiles have a strikingly similar shape, increasing during the first half of S phase then decreasing before its end. A previously proposed minimal model of stochastic initiation modulated by accumulation of a recyclable, limiting replication-fork factor and fork-promoted initiation of new origins, quantitatively described the observed profiles without requiring new implementations.The selective pressure for timely completion of genome replication and optimal usage of replication proteins that must be imported into the cell nucleus can explain the generic shape of the profiles. We have identified a universal behavior of eukaryotic replication initiation that transcends the mechanisms of origin specification. The population-averaged efficiency of replication origin usage changes during S phase in a strikingly similar manner in a highly diverse set of eukaryotes. The quantitative model previously proposed for origin activation in X. laevis can be generalized to explain this evolutionary conservation.

GC content and recombination: reassessing the causal effects for the Saccharomyces cerevisiae genome.

Recombination plays a crucial role in the evolution of genomes. Among many chromosomal features, GC content is one of the most prominent variables that appear to be highly correlated with recombination. However, it is not yet clear (1) whether recombination drives GC content (as proposed, for example, in the biased gene conversion model) or the converse and (2) what are the length scales for mutual influences between GC content and recombination. Here we have reassessed these questions for the model genome Saccharomyces cerevisiae, for which the most refined recombination data are available. First, we confirmed a strong correlation between recombination rate and GC content at local scales (a few kilobases). Second, on the basis of alignments between S. cerevisiae, S. paradoxus, and S. mikatae sequences, we showed that the inferred AT/GC substitution patterns are not correlated with recombination, indicating that GC content is not driven by recombination in yeast. These results thus suggest that, in S. cerevisiae, recombination is determined either by the GC content or by a third parameter, also affecting the GC content. Third, we observed long-range correlations between GC and recombination for chromosome III (for which such correlations were reported experimentally and were the model for many structural studies). However, similar correlations were not detected in the other chromosomes, restraining thus the generality of the phenomenon. These results pave the way for further analyses aimed at the detailed untangling of drives involved in the evolutionary shaping of the yeast genome.

DNA damage checkpoint inactivation: adaptation and recovery.

The DNA damage checkpoint is a stress response pathway detecting pathological structures of nuclear DNA and inducing appropriate responses. These responses include cell cycle arrests, histone modifications, changes in the transcription programme and post-translational modifications of proteins involved in DNA repair. Inactivation of the DNA damage checkpoint responses can occur under two circumstances: either DNA damage has disappeared and the whole pathway is inactivated in a process termed recovery, or DNA damage persists but all or part of the pathway is nevertheless inactivated, which is called adaptation. We present here a review of these inactivating processes of the DNA damage checkpoint primarily in the budding yeast Saccharomyces cerevisiae but also with reference to studies in Schizosaccharomyces pombe and in animal cells.

The checkpoint Saccharomyces cerevisiae Rad9 protein contains a tandem tudor domain that recognizes DNA.

DNA damage checkpoints are signal transduction pathways that are activated after genotoxic insults to protect genomic integrity. At the site of DNA damage, 'mediator' proteins are in charge of recruiting 'signal transducers' to molecules 'sensing' the damage. Budding yeast Rad9, fission yeast Crb2 and metazoan 53BP1 are presented as mediators involved in the activation of checkpoint kinases. Here we show that, despite low sequence conservation, Rad9 exhibits a tandem tudor domain structurally close to those found in human/mouse 53BP1 and fission yeast Crb2. Moreover, this region is important for the resistance of Saccharomyces cerevisiae to different genotoxic stresses. It does not mediate direct binding to a histone H3 peptide dimethylated on K79, nor to a histone H4 peptide dimethylated on lysine 20, as was demonstrated for 53BP1. However, the tandem tudor region of Rad9 directly interacts with single-stranded DNA and double-stranded DNAs of various lengths and sequences through a positively charged region absent from 53BP1 and Crb2 but present in several yeast Rad9 homologs. Our results argue that the tandem tudor domains of Rad9, Crb2 and 53BP1 mediate chromatin binding next to double-strand breaks. However, their modes of chromatin recognition are different, suggesting that the corresponding interactions are differently regulated.

20S proteasome assembly is orchestrated by two distinct pairs of chaperones in yeast and in mammals.

The 20S proteasome is the catalytic core of the 26S proteasome, a central enzyme in the ubiquitin-proteasome system. Its assembly proceeds in a multistep and orderly fashion. Ump1 is the only well-described chaperone dedicated to the assembly of the 20S proteasome in yeast. Here, we report a phenotype related to the DNA damage response that allowed us to isolate four other chaperones of yeast 20S proteasomes, which we named Poc1-Poc4. Poc1/2 and Poc3/4 form two pairs working at different stages in early 20S proteasome assembly. We identify PAC1, PAC2, the recently described PAC3, and an uncharacterized protein that we named PAC4 as functional mammalian homologs of yeast Poc factors. Hence, in yeast as in mammals, proteasome assembly is orchestrated by two pairs of chaperones acting upstream of the half-proteasome maturase Ump1. Our findings provide evidence for a remarkable conservation of a pairwise chaperone-assisted proteasome assembly throughout evolution.

Visualization of ribonucleotide reductase catalytic oxidation establishes thioredoxins as its major reductants in yeast.

Thioredoxins and/or glutaredoxins assist ribonucleotide reductase, and other such enzymes that require disulfide bond reduction during their catalytic cycle. In Saccharomyces cerevisiae, the presence of either pathway is essential but which of these pathways operates in ribonucleotide reductase reduction and how this function contributes to the pathways' essential nature have not been definitively established. We have identified two in vivo redox forms of the S. cerevisiae ribonucleotide reductase R1 subunit, which correspond to catalytically reduced or oxidized enzymes. Cells lacking thioredoxins, which exhibit an elongated S phase, accumulate R1 in its oxidized form and also contain significantly decreased deoxyribonucleotide levels during the S phase. Overexpressing R1 in these cells increases both the amount of the R1 reduced form and the concentrations of deoxyribonucleotides and accelerates DNA replication. These results establish thioredoxins as the major RNR reducing system in yeast and indicate that impaired RNR reduction accounts for the S phase defects of thioredoxin-deficient cells.

Mechanisms of checkpoint kinase Rad53 inactivation after a double-strand break in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, double-strand breaks (DSBs) activate DNA checkpoint pathways that trigger several responses including a strong G(2)/M arrest. We have previously provided evidence that the phosphatases Ptc2 and Ptc3 of the protein phosphatase 2C type are required for DNA checkpoint inactivation after a DSB and probably dephosphorylate the checkpoint kinase Rad53. In this article we have investigated further the interactions between Ptc2 and Rad53. We showed that forkhead-associated domain 1 (FHA1) of Rad53 interacts with a specific threonine of Ptc2, T376, located outside its catalytic domain in a TXXD motif which constitutes an optimal FHA1 binding sequence in vitro. Mutating T376 abolishes Ptc2 interaction with the Rad53 FHA1 domain and results in adaptation and recovery defects following a DSB. We found that Ckb1 and Ckb2, the regulatory subunits of the protein kinase CK2, are necessary for the in vivo interaction between Ptc2 and the Rad53 FHA1 domain, that Ckb1 binds Ptc2 in vitro and that ckb1Delta and ckb2Delta mutants are defective in adaptation and recovery after a DSB. Our data thus strongly suggest that CK2 is the kinase responsible for the in vivo phosphorylation of Ptc2 T376.

The spindle assembly checkpoint regulates the phosphorylation state of a subset of DNA checkpoint proteins in Saccharomyces cerevisiae.

The DNA and the spindle assembly checkpoints play key roles in maintaining genomic integrity by coordinating cell responses to DNA lesions and spindle dysfunctions, respectively. These two surveillance pathways seem to operate mostly independently of one another, and little is known about their potential physiological connections. Here, we show that in Saccharomyces cerevisiae, the activation of the spindle assembly checkpoint triggers phosphorylation changes in two components of the DNA checkpoint, Rad53 and Rad9. These modifications are independent of the other DNA checkpoint proteins and are abolished in spindle checkpoint-defective mutants, hinting at specific functions for Rad53 and Rad9 in the spindle damage response. Moreover, we found that after UV irradiation, Rad9 phosphorylation is altered and Rad53 inactivation is accelerated when the spindle checkpoint is activated, which suggests the implication of the spindle checkpoint in the regulation of the DNA damage response.

Gene essentiality and the topology of protein interaction networks.

The mechanistic bases for gene essentiality and for cell mutational resistance have long been disputed. The recent availability of large protein interaction databases has fuelled the analysis of protein interaction networks and several authors have proposed that gene dispensability could be strongly related to some topological parameters of these networks. However, many results were based on protein interaction data whose biases were not taken into account. In this article, we show that the essentiality of a gene in yeast is poorly related to the number of interactants (or degree) of the corresponding protein and that the physiological consequences of gene deletions are unrelated to several other properties of proteins in the interaction networks, such as the average degrees of their nearest neighbours, their clustering coefficients or their relative distances. We also found that yeast protein interaction networks lack degree correlation, i.e. a propensity for their vertices to associate according to their degrees. Gene essentiality and more generally cell resistance against mutations thus seem largely unrelated to many parameters of protein network topology.