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 MutLβ 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 MutLβ preferentially recognize D-loops, providing a mechanism for limiting gene conversion in vivo. These findings show that MutLβ 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.DOI: http://dx.doi.org/10.7554/eLife.21900.001

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.

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.

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.

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 ckb1⌬ and ckb2⌬ 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 DNA checkpoint is a surveillance mechanism that detects DNA lesions or replication blocks and coordinates various responses such as cell cycle arrests and transcriptional or posttranscriptional modifications. This mechanism is present in all eukaryotes and has been particularly analyzed in the yeast Saccharomyces cerevisiae, where it was originally identified (14, 53). In S. cerevisiae, activation of the DNA checkpoint by DNA lesions depends essentially on two sets of proteins, Rad24 and the PCNA-like trimer Rad17-Mec3-Ddc1, on the one hand, and the ATR homolog, the phosphatidylinositol 3-kinase-like Mec1 (in complex with an auxiliary subunit Ddc2), on the other hand (reviewed in references 28 and 58). Both the Rad17-Mec3-Ddc1 and the Mec1-Ddc2 complexes have been shown to be simultaneously and independently recruited to a double-strand break (DSB) artificially induced by the HO endonuclease (15,29). Once activated, Mec1 induces the phosphorylation and the activation of two central transducers, the Rad53 and Chk1 kinases, which subsequently phosphorylate downstream effectors. The phosphorylation of Rad53 and Chk1 also depends on so-called "adaptors," Rad9 in the case of DNA damage and Mrc1 in the case of replication blocks and DNA lesions during S phase (for a review on Rad53 activation, see reference 33).Rad53 plays a central part in S. cerevisiae DNA checkpoint: it controls the majority of the DNA damage responses and rad53⌬ cells are strongly hypersensitive to all genotoxic stresses. Rad53 is the founding member of the conserved family of FHA (forkhead associated) domain-containing checkpoint kinases, which also includes mammalian Chk2 and Schizosaccharomyces pombe Cds1. It contains two FHA domains, FHA1 and FHA2, flanking the protein catalytic domain. FHA domains are protein-protein interaction domains that specifica...

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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.

20 21Meiotic recombination is essential for fertility and allelic shuffling. Canonical 22 recombination models fail to capture the observed complexity of meiotic 23 recombinants. Here we revisit these models by analyzing meiotic heteroduplex DNA 24 tracts genome-wide in combination with meiotic DNA double-strand break (DSB) 25 locations. We provide unprecedented support to the synthesis-dependent strand 26 annealing model and establish estimates of its associated template switching 27 frequency and polymerase processivity. We show that resolution of double Holliday 28 junctions (dHJs) is biased toward cleavage of the pair of strands containing newly 29 synthesized DNA near the junctions. The suspected dHJ resolvase Mlh1-3 as well as 30Mlh1-2, Exo1 and Sgs1 promote asymmetric positioning of crossover intermediates 31 relative to the initiating DSB and bidirectional conversions. Finally, we show that 32 crossover-biased dHJ resolution depends on Mlh1-3, Exo1, Msh5 and to a lesser 33 extent on Sgs1. These properties are likely conserved in eukaryotes containing the 34 ZMM proteins, which includes mammals. 35