The maintenance of genome integrity and the generation of biological diversity are important biological processes, and both involve homologous recombination. In yeast and animals, homologous recombination requires the function of the RAD51 recombinase. In vertebrates, RAD51 seems to have acquired additional functions in the maintenance of genome integrity, and rad51 mutations cause lethality, but it is not clear how widely these functions are conserved among eukaryotes. We report here a loss-of-function mutant in the Arabidopsis homolog of RAD51, AtRAD51. The atrad51-1 mutant exhibits normal vegetative and flower development and has no detectable abnormality in mitosis. Therefore, AtRAD51 is not necessary under normal conditions for genome integrity. In contrast, atrad51-1 is completely sterile and defective in male and female meioses. During mutant prophase I, chromosomes fail to synapse and become extensively fragmented. Chromosome fragmentation is suppressed by atspo11-1, indicating that AtRAD51 functions downstream of AtSPO11-1. Therefore, AtRAD51 likely plays a crucial role in the repair of DNA doublestranded breaks generated by AtSPO11-1. These results suggest that RAD51 function is essential for chromosome pairing and synapsis at early stages in meiosis in Arabidopsis. Furthermore, major aspects of meiotic recombination seem to be conserved between yeast and plants, especially the fact that chromosome pairing and synapsis depend on the function of SPO11 and RAD51.H omologous recombination and DNA-damage repair are fundamental biological processes found in all life forms. Homologous recombination plays a major role in both maintaining genome stability (DNA-damage repair) and the generation of genetic variability. Defects in DNA-damage repair generally lead to genome instability and are increasingly found to be associated with cancer in mammals. The active surveillance mechanisms that can recognize and precisely repair DNA damage to prevent the accumulation of errors are meanwhile thought to be intimately involved in the prevention of cancer and the delaying of aging (1, 2). Genes playing critical roles in homologous recombination are important for these processes.Homologous recombination has been intensively studied in budding yeast Saccharomyces cerevisiae, and a number of genes have been identified that function in this process. Some of these genes, including RAD51, were identified based on the hypersensitivity of their mutants to radiation (3). RAD51 and another yeast gene, DMC1, share significant sequence homology with the bacterial recA gene (4). Similar to the bacterial RecA protein, the yeast RAD51 protein acts in homology searching, DNA pairing, and strand exchange (5), activities important for both DNAdamage repair and meiosis. RAD51 homologs have been found in all eukaryotic organisms thus far and are well studied in the vertebrates human, mouse, and chicken. In contrast to yeast, the loss of RAD51 function is lethal in both chicken DT40 and mouse cells (4). These RAD51-deficient cells arrest dur...
Interactions between homologs in meiotic prophase I, such as recombination and synapsis, are critical for proper homolog segregation and involve the coordination of several parallel events. However, few regulatory genes have been identi®ed; in particular, it is not clear what roles the proteins similar to the mitotic cell cycle regulators might play during meiotic prophase I. We describe here the isolation and characterization of a new Arabidopsis mutant called solo dancers that exhibits a severe defect in homolog synapsis, recombination and bivalent formation in meiotic prophase I, subsequently resulting in seemingly random chromosome distribution and formation of abnormal meiotic products. We further demonstrate that the mutation affects a meiosis-speci®c gene encoding a novel protein of 578 amino acid residues with up to 31% amino acid sequence identity to known cyclins in the C-terminal portion. These results argue strongly that homolog interactions during meiotic prophase I require a novel meiosis-speci®c cyclin in Arabidopsis.
Diverse kinesin motor proteins are involved in spindle function; however, the mechanisms by which they are targeted to specific sites within spindles are not well understood. Here, we show that a fusion between yellow fluorescent protein (YFP) and a minus-end-directed Kinesin-14 (C-terminal family) from Arabidopsis, ATK5, localizes to mitotic spindle midzones and regions rich in growing plus-ends within phragmoplasts. Notably, in Arabidopsis interphase cells, YFP::ATK5 localizes to microtubules with a preferential enrichment at growing plus-ends; indicating ATK5 is a plus-end tracking protein (؉TIP). This ؉TIP activity is conferred by regions outside of the C-terminal motor domain, which reveals the presence of independent plus-end tracking and minus-end motor activities within ATK5. Furthermore, mitotic spindles of atk5 null mutant plants are abnormally broadened. Based on these data, we propose a model in which ATK5 uses plus-end tracking to reach spindle midzones, where it then organizes microtubules via minus-end-directed motor activity. INTRODUCTIONDuring cell division, the proper segregation of genetic material into daughter cells requires the action of the microtubule spindle apparatus and its associated proteins. The spindle consists of two opposing sets of microtubules oriented with the minus-ends at the poles and the plus-ends at the midzone. The midzone represents the region of overlap between the two halves of the spindle, where microtubule plus-ends terminate at chromosomal kinetochores (kinetochore microtubules) or interdigitate in an antiparallel manner with microtubules from the opposite pole (interpolar microtubules). The spindle midzone is the site of force generation during anaphase spindle elongation (Leslie and Pickett-Heaps, 1983;Khodjakov et al., 2004), and in plants it persists through telophase to form the cytokinetic microtubule apparatus, the phragmoplast (Euteneuer et al., 1982).The assembly and functioning of spindles involve the highly orchestrated activities of diverse microtubule motor proteins. Kinesins convert the energy derived from ATP hydrolysis into translational movement along microtubules (Dagenbach and Endow, 2004;Lawrence et al., 2004). Kinesin-14 family members (previously referred to as C-terminal kinesins), such as Ncd from Drosophila and Kar3p from budding yeast, are unique in that they translocate exclusively toward microtubule minus-ends (Walker et al., 1990). Several Kinesin-14 family members contain microtubule binding sites in their tail regions, which specifies the ability to carry microtubules as cargo along other microtubules; in effect, moving microtubules in relation to one another (Walczak et al., 1997;Narasimhulu and Reddy, 1998;Karabay and Walker, 1999;Matuliene et al., 1999). This finding, in conjunction with subcellular localization and loss-of-function studies, has revealed two distinct roles for Kinesin-14s in spindle functioning. The first role is inferred from studies showing that loss or depletion of various Kinesin-14 family members rescues the s...
Meiotic prophase I is a complex process involving homologous chromosome (homolog) pairing, synapsis, and recombination. The budding yeast (Saccharomyces cerevisiae) RAD51 gene is known to be important for recombination and DNA repair in the mitotic cell cycle. In addition, RAD51 is required for meiosis and its Arabidopsis (Arabidopsis thaliana) ortholog is important for normal meiotic homolog pairing, synapsis, and repair of double-stranded breaks. In vertebrate cell cultures, the RAD51 paralog RAD51C is also important for mitotic homologous recombination and maintenance of genome integrity. However, the function of RAD51C in meiosis is not well understood. Here we describe the identification and analysis of a mutation in the Arabidopsis RAD51C ortholog, AtRAD51C. Although the atrad51c-1 mutant has normal vegetative and flower development and has no detectable abnormality in mitosis, it is completely male and female sterile. During early meiosis, homologous chromosomes in atrad51c-1 fail to undergo synapsis and become severely fragmented. In addition, analysis of the atrad51c-1 atspo11-1 double mutant showed that fragmentation was nearly completely suppressed by the atspo11-1 mutation, indicating that the fragmentation largely represents a defect in processing double-stranded breaks generated by AtSPO11-1. Fluorescence in situ hybridization experiments suggest that homolog juxtaposition might also be abnormal in atrad51c-1 meiocytes. These results demonstrate that AtRAD51C is essential for normal meiosis and is probably required for homologous synapsis.Meiosis is essential for eukaryotic sexual reproduction, allowing the production of haploid gametes. In addition, meiotic recombination during the early stages of meiosis allows the exchange of genetic information, serving as an important source of genetic diversity. The success of meiosis depends on a complex and prolonged prophase I that involves homologous chromosome (homolog) pairing, synapsis, and recombination (Zickler and Kleckner, 1999;Page and Hawley, 2003;Schwarzacher, 2003). After pairing, the homologs continue to associate and this interaction has been referred to as homolog juxtaposition (Zickler and Kleckner, 1999). Recombination results in crossover events that correspond to cytologically observed chiasmata, which, in combination with sister chromatid cohesion, maintain the association between homologs in the form of bivalents, ensuring proper segregation of homologs at anaphase I. Synapsis, the formation of synaptonemal complexes (SCs) between closely associated chromosomes, has also been implicated to play important roles in meiotic prophase I, although its relationship with recombination differs among organisms.Cytological and molecular genetic studies support the idea that homolog pairing, synapsis, and recombination are closely coupled events in normal meiosis. In particular, recombination and synapsis are often interdependent. In fact, a number of meiotic genes are required for both normal synapsis and recombination in yeast (Saccharomyces cer...
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