Meiotic recombination and crossovers in plants.

Efforts have been made in recent years to clarify molecular meiotic processes in a large variety of higher eukaryotes. In plants, such studies have enjoyed a boom in the last years with the use of Arabidopsis thaliana together with maize, rice and tomato as model systems. Owing to direct and reverse genetic screens, an increasing number of genes involved in meiosis have been characterized in plants. In parallel, the improvement of cytological and genetical tools has allowed a precise description of meiotic recombination events. Thus, it appears that meiotic studies in plants are reaching a new stage and can provide new insights into meiotic recombination mechanisms. In this review, we intend to give an overview of these recent advances in the understanding of meiotic recombination in plants.

Patterns of recombination and MLH1 foci density along mouse chromosomes: modeling effects of interference and obligate chiasma.

Crossover interference in meiosis is often modeled via stationary renewal processes. Here we consider a new model to incorporate the known biological feature of "obligate chiasma" whereby in most organisms each bivalent almost always has at least one crossover. The initial crossover is modeled as uniformly distributed along the chromosome, and starting from its position, subsequent crossovers are placed with forward and backward stationary renewal processes using a chi-square distribution of intercrossover distances. We used our model as well as the standard chi-square model to simulate the patterns of crossover densities along bivalents or chromatids for those having zero, one, two, or three or more crossovers; indeed, such patterns depend on the number of crossovers. With both models, simulated patterns compare very well to those found experimentally in mice, both for MLH1 foci on bivalents and for crossovers on genetic maps. However, our model provides a better fit to experimental data as compared to the standard chi-square model, particularly regarding the distribution of numbers of crossovers per chromosome. Finally, our model predicts an enhancement of the recombination rate near the extremities, which, however, explains only a part of the pattern observed in mouse.

Meiotic recombination hotspots in plants.

Many studies have demonstrated that the distribution of meiotic crossover events along chromosomes is non-random in plants and other species with sexual reproduction. Large differences in recombination frequencies appear at several scales. On a large scale, regions of high and low rates of crossover have been found to alternate along the chromosomes in all plant species studied. High crossover rates have been reported to be correlated with several chromosome features (e.g. gene density and distance to the centromeres). However, most of these correlations cannot be extended to all plant species. Only a few plant species have been studied on a finer scale. Hotspots of meiotic recombination (i.e. DNA fragments of a few kilobases in length with a higher rate of recombination than the surrounding DNA) have been identified in maize and rice. Most of these hotspots are intragenic. In Arabidopsis thaliana, we have identified several DNA fragments (less than 5 kb in size) with genetic recombination rates at least 5 times higher than the whole-chromosome average [4.6 cM (centimorgan)/Mb], which are therefore probable hotspots for meiotic recombination. Most crossover breakpoints lie in intergenic or non-coding regions. Major efforts should be devoted to characterizing meiotic recombination at the molecular level, which should help to clarify the role of this process in genome evolution.

Helicase-defective RuvB(D113E) promotes RuvAB-mediated branch migration in vitro.

In Escherichia coli, the RuvA and RuvB proteins interact at Holliday junctions to promote branch migration leading to the formation of heteroduplex DNA. RuvA provides junction-binding specificity and RuvB drives ATP-dependent branch migration. Since RuvB contains sequence motifs characteristic of a DNA helicase and RuvAB exhibit helicase activity in vitro, we have analysed the role of DNA unwinding in relation to branch migration. A mutant RuvB protein, RuvB(D113E), mutated in helicase motif II (the DExx box), has been purified to homogeneity. The mutant protein forms hexameric rings on DNA similar to those formed by wild-type protein and promotes branch migration in the presence of RuvA. However, RuvB(D113E) exhibits reduced ATPase activity and is severely compromised in its DNA helicase activity. Models for RuvAB-mediated branch migration that invoke only limited DNA unwinding activity are proposed.

Escherichia coli RuvBL268S: a mutant RuvB protein that exhibits wild-type activities in vitro but confers a UV-sensitive ruv phenotype in vivo.

The RuvABC proteins of Escherichia coli process recombination intermediates during genetic recombination and DNA repair. RuvA and RuvB promote branch migration of Holliday junctions, a process that extends heteroduplex DNA. Together with RuvC, they form a RuvABC complex capable of Holliday junction resolution. Branch migration by RuvAB is mediated by RuvB, a hexameric ring protein that acts as an ATP-driven molecular pump. To gain insight into the mechanism of branch migration, random mutations were introduced into the ruvB gene by PCR and a collection of mutant alleles were obtained. Mutation of leucine 268 to serine resulted in a severe UV-sensitive phenotype, characteristic of a ruv defect. Here, we report a biochemical analysis of the mutant protein RuvBL268S. Unexpectedly, the purified protein is fully active in vitro with regard to its ATPase, DNA binding and DNA unwinding activities. It also promotes efficient branch migration in combination with RuvA, and forms functional RuvABC-Holliday junction resolvase complexes. These results indicate that RuvB may perform some additional, and as yet undefined, function that is necessary for cell survival after UV-irradiation.

[Mechanisms and control of meiotic recombination in the yeast Saccharomyces cerevisiae]. / Mécanismes et contrôles de la recombinaison méiotique chez la levure Saccharomyces cerevisiae.

Recent studies in Saccharomyces cerevisiae have provided new insights in our understanding of the molecular mechanisms of meiotic recombination. Meiosis-specific DNA double-strand breaks have been detected and have been shown to be the lesions that initiate recombination events. These are located mostly in promoter regions where the chromatin is in an open configuration, and cluster in domains along the chromosome. They are likely to be made by a topoisomerase II-like protein encoded by the SPO11 gene. Several DNA intermediates in the meiotic double strand-break repair pathway have been characterised and several multi-protein complexes have been identified and shown to be involved at different steps in the repair pathway. The conservation of these protein complexes in higher eukaryotes suggests that the meiotic recombination mechanism could be conserved. With the application of the well characterised genetical, molecular, cytological and biochemical techniques and the recently developed technology for genomic studies (biochips), we can expect a rapid increase in our comprehension of the meiotic recombination process.

Coordinated actions of RuvABC in Holliday junction processing.

The RuvA, RuvB and RuvC proteins of Escherichia coli process Holliday junctions during genetic recombination and DNA repair. Biochemical studies have shown that RuvA and RuvB promote branch migration whereas RuvC resolves junctions by endonucleolytic cleavage. Here we show that RuvAB stimulate Holliday junction resolution by RuvC. Elevated RuvC activity was dependent upon RuvAB-mediated ATP-hydrolysis. These results show that the three Ruv proteins work in a coordinated manner to promote Holliday junction resolution, and account for the resolvase-defective phenotype exhibited by ruvA, ruvB or ruvC mutant strains.

Functional interactions between the holliday junction resolvase and the branch migration motor of Escherichia coli.

Homologous recombination generates genetic diversity and provides an important cellular pathway for the repair of double-stranded DNA breaks. Two key steps in this process are the branch migration of Holliday junctions followed by their resolution into mature recombination products. In E.coli, branch migration is catalysed by the RuvB protein, a hexameric DNA helicase that is loaded onto the junction by RuvA, whereas resolution is promoted by the RuvC endonuclease. Here we provide direct evidence for functional interactions between RuvB and RuvC that link these biochemically distinct processes. Using synthetic Holliday junctions, RuvB was found to stabilize the binding of RuvC to a junction and to stimulate its resolvase activity. Conversely, RuvC facilitated interactions between RuvB and the junction such that RuvBC complexes catalysed branch migration. The observed synergy between RuvB and RuvC provides new insight into the structure and function of a RuvABC complex that is capable of facilitating branch migration and resolution of Holliday junctions via a concerted enzymatic mechanism.

Biochemical properties of RuvBD113N: a mutation in helicase motif II of the RuvB hexamer affects DNA binding and ATPase activities.

Many DNA helicases utilise the energy derived from nucleoside triphosphate hydrolysis to fuel their actions as molecular motors in a variety of biological processes. In association with RuvA, the E. coli RuvB protein (a hexameric ring helicase), promotes the branch migration of Holliday junctions during genetic recombination and DNA repair. To analyse the relationship between ATP-dependent DNA helicase activity and branch migration, a site-directed mutation was introduced into the helicase II motif of RuvB. Over-expression of RuvBD113N in wild-type E. coli resulted in a dominant negative UVs phenotype. The biochemical properties of RuvBD113N were examined and compared with wild-type RuvB in vitro. The single amino acid substitution resulted in major alterations to the biochemical activities of RuvB, such that RuvBD113N was defective in DNA binding and ATP hydrolysis, while retaining the ability to form hexameric rings and interact with RuvA. RuvBD113N formed heterohexamers with wild-type RuvB, and could inhibit RuvB function by affecting its ability to bind DNA. However, heterohexamers exhibited an ability to promote branch migration in vitro indicating that not all subunits of the ring need to be catalytically competent.

Sequence comparison of the ARG4 chromosomal regions from the two related yeasts, Saccharomyces cerevisiae and Saccharomyces douglasii.

A 3.6 kb DNA fragment from Saccharomyces douglasii, containing the ARG4 gene, has been cloned, sequenced and compared to the corresponding region from Saccharomyces cerevisiae. The organization of this region is identical in both yeasts. It contains besides the ARG4 gene, another complete open reading frame (ORF) (YSD83) and a third incomplete one (DED81). The ARG4 and the YSD83 coding regions differ from their S. cerevisiae homologs by 8.1% and 12.5%, respectively, of base substitutions. The encoded proteins have evolved differently: amino acid replacements are significantly less frequent in Arg4 (2.8%) than in Ysc83 (12.4%) and most of the changes in Arg4 are conservative, which is not the case for Ysc83. The non-coding regions are less conserved, with small AT-rich insertions/deletions and 20% base substitutions. However, the level of divergence is smaller in the aligned sequences of these regions than in silent sites of the ORFs, probably revealing a higher degree of constraints. The Gcn4 binding site and the region where meiotic double-strand breaks occur, are fully conserved. The data confirm that these two yeasts are evolutionarily closely related and that comparisons of their sequences might reveal conserved protein and DNA domains not expected to be found in sequence comparisons between more diverged organisms.

Homologous, homeologous, and illegitimate repair of double-strand breaks during transformation of a wild-type strain and a rad52 mutant strain of Saccharomyces cerevisiae.

Different modes of in vivo repair of double-strand breaks (DSBs) have been described for various organisms: the recombinational DSB repair (DSBR) mode, the single-strand annealing (SSA) mode, and end-to-end joining. To investigate these modes of DSB repair in Saccharomyces cerevisiae, we have examined the fate of in vitro linearized replicative plasmids during transformation with respect to several parameters. We found that (i) the efficiencies of both intramolecular and intermolecular linear plasmid DSB repair are homology dependent (according to the amount of DNA used during transformation [100 ng or less], recombination between similar but not identical [homeologous] P450s sequences sharing 73% identity is 2- to 18-fold lower than recombination between identical sequences); (ii) the RAD52 gene product is not essential for intramolecular recombination between homologous and homeologous direct repeats (as in the wild-type strain, recombination occurs with respect to the overall alignment of the parental sequences); (iii) in contrast, the RAD52 gene product is required for intermolecular interactions (the rare transformants which are obtained contain plasmids resulting from deletion-forming intramolecular events involving little or no sequence homology); (iv) similarly, sequencing data revealed examples of intramolecular joining within the few terminal nucleotides of the transforming DNA upon transformation with a linear plasmid with no repeat in the wild-type strain. The recombinant junctions of the rare illegitimate events obtained with S. cerevisiae are very similar to those observed in the repair of DSB in mammalian cells. Together, these and previous results suggest the existence of alternative modes for DSB repair during transformation which differ in their efficiencies and in the structure of their products. We discuss the implications of these results with respect to the existence of alternative pathways and the role of the RAD52 gene product.

Recombination between similar but not identical DNA sequences during yeast transformation occurs within short stretches of identity.

Interactions between similar but not identical (homeologous) DNA sequences play an important biological role in the evolution of genes and genomes. To gain insight into the underlying molecular mechanism(s) of genetic recombination, we have studied inter- and intramolecular homeologous recombination in S. cerevisiae during transformation. We found that homeologous DNAs recombine efficiently. Hybrid sequences were obtained between two mammalian cytochrome P450 cDNAs, sharing 73% identity, and between the yeast ARG4 gene and its human homeologous cDNA, sharing 52% identity. Sequencing data showed that the preferred recombination events are those corresponding to the overall alignment of the DNA sequences and that the junctions are within stretches of identity of variable length (2-21 nt). We suggest that these events occur by a conventional homologous recombination mechanism.

A "G" to "A" mutation at position -1 of a 5' splice site in a late infantile form of Tay-Sachs disease.

Tay-Sachs disease is an autosomal recessive genetic disease caused by a deficiency in beta-hexosaminidase A. We have characterized a new mutation in a Tunisian patient displaying a late infantile form of Tay-Sachs disease. Northern blot analysis of patient's fibroblast total RNAs shows a broad, fast migrating band in the region of the normal beta-hexosaminidase alpha transcripts. The mRNA coding for beta-hexosaminidase alpha subunit was first reverse transcribed and then amplified in four overlapping segments spanning the entire coding sequence by polymerase chain reaction. We found in the products of polymerase chain reaction (PCR) that amplify the segment spanning exons 2-7, in addition to a normal fragment, two smaller size fragments, one of which is also seen in normal control fibroblasts. The analysis of the patient's specific abnormal fragment by hybridization with exon-specific oligonucleotides and then sequencing allowed us to conclude that this fragment lacked exon 5. The other smaller species lacked exons 4 and 5 in both patient and normal control. The sequence of a genomic fragment containing exon 5 and of the patient's normal cDNA fragment spanning exons 2-7, revealed a point mutation G to A at the last nucleotide of exon 5. This mutation doesn't change the sense of the affected codon. Northern blot of patient's fibroblast poly(A+) RNAs allowed us to quantify two of the forms of transcripts seen by PCR. In the patient, the normal size transcript and the exon 5-deleted transcript represent, respectively, 3 and 7% of the normal control beta-hexosaminidase alpha mRNA. We propose that this point mutation is responsible for an inefficient and abnormal processing of the mutant transcript resulting in the appearance of two low abundance spliced mRNAs. One is lacking exon 5 and most likely codes for an inactive protein; the other is similar to normal beta-hexosaminidase alpha mRNA, except for the presence of the silent G to A mutation and codes therefore for a normal enzyme accounting for the 2.5% residual beta-hexosaminidase A activity measured in patient's fibroblasts by a fluorometric assay. The third form, without exons 4 and 5, is also evidenced in normal fibroblasts by PCR so that we think that it is not related to Tay-Sachs disease.

Prenatal diagnosis of mucolipidosis type II on first-trimester amniotic fluid.

We investigated the possibility of prenatal diagnosis of mucolipidosis type II (ML II) by lysosomal enzyme determination on amniotic fluid obtained at 11 weeks of gestation in three pregnancies at risk. Diagnosis of ML II was made in one case on the basis of increased levels of five lysosomal enzymes tested. The diagnosis was confirmed on cultured chorionic cells, their cultured medium, 17-week amniotic fluid, and fetal plasma obtained for confirmation prior to the termination of pregnancy.