Characterization of Brca2-deficient plants excludes the role of NHEJ and SSA in the meiotic chromosomal defect phenotype.

In somatic cells, three major pathways are involved in the repair of DNA double-strand breaks (DBS): Non-Homologous End Joining (NHEJ), Single-Strand Annealing (SSA) and Homologous Recombination (HR). In somatic and meiotic HR, DNA DSB are 5' to 3' resected, producing long 3' single-stranded DNA extensions. Brca2 is essential to load the Rad51 recombinase onto these 3' overhangs. The resulting nucleofilament can thus invade a homologous DNA sequence to copy and restore the original genetic information. In Arabidopsis, the inactivation of Brca2 specifically during meiosis by an RNAi approach results in aberrant chromosome aggregates, chromosomal fragmentation and missegregation leading to a sterility phenotype. We had previously suggested that such chromosomal behaviour could be due to NHEJ. In this study, we show that knock-out plants affected in both BRCA2 genes show the same meiotic phenotype as the RNAi-inactivated plants. Moreover, it is demonstrated that during meiosis, neither NHEJ nor SSA compensate for HR deficiency in BRCA2-inactivated plants. The role of the plant-specific DNA Ligase6 is also excluded. The possible mechanism(s) involved in the formation of these aberrant chromosomal bridges in the absence of HR during meiosis are discussed.

Homologous recombination is stimulated by a decrease in dUTPase in Arabidopsis.

Deoxyuridine triphosphatase (dUTPase) enzyme is an essential enzyme that protects DNA against uracil incorporation. No organism can tolerate the absence of this activity. In this article, we show that dUTPase function is conserved between E. coli (Escherichia coli), yeast (Saccharomyces cerevisiae) and Arabidopsis (Arabidopsis thaliana) and that it is essential in Arabidopsis as in both micro-organisms. Using a RNA interference strategy, plant lines were generated with a diminished dUTPase activity as compared to the wild-type. These plants are sensitive to 5-fluoro-uracil. As an indication of DNA damage, inactivation of dUTPase results in the induction of AtRAD51 and AtPARP2, which are involved in DNA repair. Nevertheless, RNAi/DUT1 constructs are compatible with a rad51 mutation. Using a TUNEL assay, DNA damage was observed in the RNAi/DUT1 plants. Finally, plants carrying a homologous recombination (HR) exclusive substrate transformed with the RNAi/DUT1 construct exhibit a seven times increase in homologous recombination events. Increased HR was only detected in the plants that were the most sensitive to 5-fluoro-uracils, thus establishing a link between uracil incorporation in the genomic DNA and HR. Our results show for the first time that genetic instability provoked by the presence of uracils in the DNA is poorly tolerated and that this base misincorporation globally stimulates HR in plants.

The SOS screen in Arabidopsis: a search for functions involved in DNA metabolism.

The SOS screen, as originally described by Perkins et al. (1999) [7], was setup with the aim of identifying Arabidopsis functions that might potentially be involved in the DNA metabolism. Such functions, when expressed in bacteria, are prone to disturb replication and thus trigger the SOS response. Consistently, expression of AtRAD51 and AtDMC1 induced the SOS response in bacteria, even affecting E. coli viability. 100 SOS-inducing cDNAs were isolated from a cDNA library constructed from an Arabidopsis cell suspension that was found to highly express meiotic genes. A large proportion of these SOS(+) candidates are clearly related to the DNA metabolism, others could be involved in the RNA metabolism, while the remaining cDNAs encode either totally unknown proteins or proteins that were considered as irrelevant. Seven SOS(+) candidate genes are induced following gamma irradiation. The in planta function of several of the SOS-inducing clones was investigated using T-DNA insertional mutants or RNA interference. Only one SOS(+) candidate, among those examined, exhibited a defined phenotype: silenced plants for DUT1 were sensitive to 5-fluoro-uracil (5FU), as is the case of the leaky dut-1 mutant in E. coli that are affected in dUTPase activity. dUTPase is essential to prevent uracil incorporation in the course of DNA replication.

Arabidopsis uracil DNA glycosylase (UNG) is required for base excision repair of uracil and increases plant sensitivity to 5-fluorouracil.

Uracil in DNA arises by misincorporation of dUMP during replication and by hydrolytic deamination of cytosine. This common lesion is actively removed through a base excision repair (BER) pathway initiated by a uracil DNA glycosylase (UDG) activity that excises the damage as a free base. UDGs are classified into different families differentially distributed across eubacteria, archaea, yeast, and animals, but remain to be unambiguously identified in plants. We report here the molecular characterization of AtUNG (Arabidopsis thaliana uracil DNA glycosylase), a plant member of the Family-1 of UDGs typified by Escherichia coli Ung. AtUNG exhibits the narrow substrate specificity and single-stranded DNA preference that are characteristic of Ung homologues. Cell extracts from atung(-/-) mutants are devoid of UDG activity, and lack the capacity to initiate BER on uracil residues. AtUNG-deficient plants do not display any apparent phenotype, but show increased resistance to 5-fluorouracil (5-FU), a cytostatic drug that favors dUMP misincorporation into DNA. The resistance of atung(-/-) mutants to 5-FU is accompanied by the accumulation of uracil residues in DNA. These results suggest that AtUNG excises uracil in vivo but generates toxic AP sites when processing abundant U:A pairs in dTTP-depleted cells. Altogether, our findings point to AtUNG as the major UDG activity in Arabidopsis.

PHS1 regulates meiotic recombination and homologous chromosome pairing by controlling the transport of RAD50 to the nucleus.

Recombination and pairing of homologous chromosomes are critical for bivalent formation in meiotic prophase. In many organisms, including yeast, mammals, and plants, pairing and recombination are intimately interconnected. The POOR HOMOLOGOUS SYNAPSIS1 (PHS1) gene acts in coordination of chromosome pairing and early recombination steps in plants, ensuring pairing fidelity and proper repair of meiotic DNA double-strand-breaks. In phs1 mutants, chromosomes exhibit early recombination defects and frequently associate with non-homologous partners, instead of pairing with their proper homologs. Here, we show that the product of the PHS1 gene is a cytoplasmic protein that functions by controlling transport of RAD50 from cytoplasm to the nucleus. RAD50 is a component of the MRN protein complex that processes meiotic double-strand-breaks to produce single-stranded DNA ends, which act in the homology search and recombination. We demonstrate that PHS1 plays the same role in homologous pairing in both Arabidopsis and maize, whose genomes differ dramatically in size and repetitive element content. This suggests that PHS1 affects pairing of the gene-rich fraction of the genome rather than preventing pairing between repetitive DNA elements. We propose that PHS1 is part of a system that regulates the progression of meiotic prophase by controlling entry of meiotic proteins into the nucleus. We also document that in phs1 mutants in Arabidopsis, centromeres interact before pairing commences along chromosome arms. Centromere coupling was previously observed in yeast and polyploid wheat while our data suggest that it may be a more common feature of meiosis.

Zip4/Spo22 is required for class I CO formation but not for synapsis completion in Arabidopsis thaliana.

In budding yeast meiosis, the formation of class I interference-sensitive crossovers requires the ZMM proteins. These ZMM proteins are essential in forming a mature synaptonemal complex, and a subset of these (Zip2, Zip3, and Zip4) has been proposed to compose the core of synapsis initiation complexes (SICs). Zip4/Spo22 functions with Zip2 to promote polymerization of Zip1 along chromosomes, making it a crucial SIC component. In higher eukaryotes, synapsis and recombination have often been correlated, but it is totally unknown how these two processes are linked. In this study, we present the characterization of a higher eukaryote SIC component homologue: Arabidopsis AtZIP4. We show that mutations in AtZIP4 belong to the same epistasis group as Atmsh4 and eliminate approximately 85% of crossovers (COs). Furthermore, genetic analyses on two adjacent intervals of Chromosome I established that the remaining COs in Atzip4 do not show interference. Lastly, immunolocalization studies showed that polymerization of the central element of the synaptonemal complex is not affected in Atzip4 background, even if it may proceed from fewer sites compared to wild type. These results reveal that Zip4 function in class I CO formation is conserved from budding yeast to Arabidopsis. On the other hand, and contrary to the situation in yeast, mutation in AtZIP4 does not prevent synapsis, showing that both aspects of the Zip4 function (i.e., class I CO maturation and synapsis) can be uncoupled.

Interaction between Arabidopsis Brca2 and its partners Rad51, Dmc1, and Dss1.

The Arabidopsis (Arabidopsis thaliana) orthologs of Brca2, a protein whose mutations are involved in breast cancer in humans, were previously shown to be essential at meiosis. In an attempt to better understand the Brca2-interacting properties, we examined four partners of the two isoforms of Brca2 identified in Arabidopsis (AtRad51, AtDmc1, and two AtDss1 isoforms). The two Brca2 and the two Dss1 isoforms are named AtBrca2(IV), AtBrca2(V), AtDss1(I), and AtDss1(V) after their chromosomal localization. We first show that both AtBrca2 proteins can interact with either AtRad51 or AtDmc1 in vitro, and that the N-terminal region of AtBrca2 is responsible for these interactions. More specifically, the BRC motifs (so called because iterated in the Brca2 protein) in Brca2 are involved in these interactions: BRC motif number 2 (BRC2) alone can interact with AtDmc1, whereas BRC motif number 4 (BRC4) recognizes AtRad51. The human Rad51 and Dmc1 proteins themselves can interact with either the complete (HsRad51) or a shorter version of AtBrca2 (HsRad51 or HsDmc1) that comprises all four BRC motifs. We also identified two Arabidopsis isoforms of Dss1, another known partner of Brca2 in other organisms. Although all four Brca2 and Dss1 proteins are much conserved, AtBrca2(IV) interacts with only one of these AtDss1 proteins, whereas AtBrca2(V) interacts with both of them. Finally, we show for the first time that an AtBrca2 protein could bind two different partners at the same time: AtRad51 and AtDss1(I), or AtDmc1 and AtDss1(I).

Two meiotic crossover classes cohabit in Arabidopsis: one is dependent on MER3,whereas the other one is not.

BACKGROUND: Crossovers are essential for the completion of meiosis. Recently, two pathways of crossover formation have been identified on the basis of distinct genetic controls. In one pathway, crossover inhibits the occurrence of another such event in a distance-dependent manner. This phenomenon is known as interference. The second kind of crossover is insensitive to interference. The two pathways function independently in budding yeast. Only interference-insensitive crossovers occur in Schizosaccharomyces pombe. In contrast, only interference-sensitive crossovers occur in Caenorabditis elegans. The situation in mammals and plants remains unclear. Mer3 is one of the genes shown to be required for the formation of interference-sensitive crossovers in Saccharomyces cerevisiae. RESULTS: To unravel the crossover status in the plant Arabidopsis thaliana, we investigated the role of the A. thaliana MER3 gene through the characterization of a series of allelic mutants. All mer3 mutants showed low levels of fertility and a significant decrease (about 75%) but not a total disappearance of meiotic crossovers, with the number of recombination events initiated in the mutants being similar to that in the wild-type. Genetic analyses showed that the residual crossovers in mer3 mutants did not display interference in one set of adjacent intervals. CONCLUSIONS: Mutation in MER3 in Arabidopsis appeared to be specific to recombination events resulting in interference-sensitive crossovers. Thus, MER3 function is conserved from yeast to plants and may exist in other metazoans. Arabidopsis therefore has at least two pathways for crossover formation, one giving rise to interference-sensitive crossover and the other to independently distributed crossovers.

Brca2 is involved in meiosis in Arabidopsis thaliana as suggested by its interaction with Dmc1.

Two BRCA2-like sequences are present in the Arabidopsis genome. Both genes are expressed in flower buds and encode nearly identical proteins, which contain four BRC motifs. In a yeast two-hybrid assay, the Arabidopsis Brca2 proteins interact with Rad51 and Dmc1. RNAi constructs aimed at silencing the BRCA2 genes at meiosis triggered a reproducible sterility phenotype, which was associated with dramatic meiosis alterations. We obtained the same phenotype upon introduction of RNAi constructs aimed at silencing the RAD51 gene at meiosis in dmc1 mutant plants. The meiotic figures we observed strongly suggest that homologous recombination is highly disturbed in these meiotic cells, leaving aberrant recombination events to repair the meiotic double-strand breaks. The 'brca2' meiotic phenotype was eliminated in spo11 mutant plants. Our experiments point to an essential role of Brca2 at meiosis in Arabidopsis. We also propose a role for Rad51 in the dmc1 context.

[Molecular mechanisms of meiosis in plants]. / Les mécanismes moléculaires de la méiose chez les plantes.

Meiosis is a key step in diploid sexual reproduction. Apart from its cytological description, the molecular mechanisms involved in this specialized cell division are being deciphered in plants thanks to the model plant Arabidopsis thaliana. While some meiotic mutants of Arabidopsis confirm the central role of functions that have been described either in yeast or in mice, others led to the identification of previously unknown genes. Numerous plants also exist as polyploids, which represent a special case with regard to meiosis.