Replication protein A (AtRPA1a) is required for class I crossover formation but is dispensable for meiotic DNA break repair

Replication protein A (RPA) is involved in many aspects of DNA metabolism including meiotic recombination. Many species possess a single RPA1 gene but Arabidopsis possesses five RPA1 paralogues. This feature has enabled us to gain further insight into the meiotic role of RPA1. Proteomic analysis implicated one of the AtRPA1 family (AtRPA1a) in meiosis. Immunofluorescence studies confirmed that AtRPA1a is associated with meiotic chromosomes from leptotene through to early pachytene. Analysis of an Atrpa1a mutant revealed that AtRPA1a is not essential at early stages in the recombination pathway. DNA double‐strand breaks are repaired in Atrpa1a, but the mutant is defective in the formation of crossovers, exhibiting a 60% reduction in chiasma frequency. Consistent with this, localization of recombination proteins AtRAD51 and AtMSH4 appears normal, whereas the numbers of AtMLH1 and AtMLH3 foci at pachytene are significantly reduced. This suggests that the defect in Atrpa1a is manifested at the stage of second‐end capture. Analysis of Atrpa1a/Atmsh4 and Atrpa1a/Atmlh3 double mutants indicates that loss of AtRPA1a predominantly affects the formation of class I, interference‐dependent crossovers.     

The Arabidopsis thaliana PARTING DANCERS gene encoding a novel protein is required for normal meiotic homologous recombination

Recent studies of meiotic recombination in the budding yeast and the model plant Arabidopsis thaliana indicate that meiotic crossovers (COs) occur through two genetic pathways: the interference-sensitive pathway and the interference-insensitive pathway. However, few genes have been identified in either pathway. Here, we describe the identification of the PARTING DANCERS (PTD) gene, as a gene with an elevated expression level in meiocytes. Analysis of two independently generated transferred DNA insertional lines in PTD showed that the mutants had reduced fertility. Further cytological analysis of male meiosis in the ptd mutants revealed defects in meiosis, including reduced formation of chiasmata, the cytological appearance of COs. The residual chiasmata in the mutants were distributed randomly, indicating that the ptd mutants are defective for CO formation in the interference-sensitive pathway. In addition, transmission electron microscopic analysis of the mutants detected no obvious abnormality of synaptonemal complexes and apparently normal late recombination nodules at the pachytene stage, suggesting that the mutant's defects in bivalent formation were postsynaptic. Comparison to other genes with limited sequence similarity raises the possibility that PTD may present a previously unknown function conserved in divergent eukaryotic organisms.     

The fission yeast pfh1(+) gene encodes an essential 5' to 3' DNA helicase required for the completion of S-phase

The Cdc24 protein plays an essential role in chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe, most likely via its direct interaction with Dna2, a conserved endonuclease–helicase protein required for Okazaki fragment processing. To gain insights into Cdc24 function, we isolated cold-sensitive chromosomal suppressors of the temperature-sensitive cdc24-M38 allele. One of the complementation groups of such suppressors defined a novel gene, pfh1⁺, encoding an 805 amino acid nuclear protein highly homologous to the Saccharomyces cerevisiae Pif1p and Rrm3p DNA helicase family proteins. The purified Pfh1 protein displayed single-stranded DNA-dependent ATPase activity as well as 5′ to 3′ DNA helicase activity in vitro. Reverse genetic analysis in S.pombe showed that helicase activity was essential for the function of the Pfh1 protein in vivo. Schizosaccharomyces pombe cells carrying the cold-sensitive pfh1-R20 allele underwent cell cycle arrest in late S/G2-phase of the cell cycle when shifted to the restrictive temperature. This arrest was dependent upon the presence of a functional late S/G2 DNA damage checkpoint, suggesting that Pfh1 is required for the comple tion of DNA replication. Furthermore, at their permissive temperature pfh1-R20 cells were highly sensitive to the DNA-alkylating agent methyl methanesulphonate, implying a further role for Pfh1 in the repair of DNA damage.     

Characterization of REC104, a gene required for early meiotic recombination in the yeast Saccharomyces cerevisiae

The REC104 gene was initially defined by mutations that rescued the inviability of a rad52 spo13 haploid strain in meiosis. We have observed that rec104 mutant strains undergo essentially no induction of meiotic gene conversion, and we have not been able to detect any meiotic crossing over in such strains. The REC104 gene has no apparent role in mitosis, since mutations have no observable effect on growth, mitotic recombination, or DNA repair. The DNA sequence of REC104 reveals that it is a previously unknown gene with a coding region of 549-bp, and genetic mapping has localized the gene to chromosome VIll near FUR1. Expression of the REC104 gene is induced in meiosis, and it appears that the gene is not transcribed in mitotic cells. Possible roles for the REC104 gene product in meiosis are discussed. © 1993 Wiley-Liss, Inc.     

The AP-3 adaptor complex is required for vacuolar function in Arabidopsis

Subcellular trafficking is required for a multitude of functions in eukaryotic cells. It involves regulation of cargo sorting, vesicle formation, trafficking and fusion processes at multiple levels. Adaptor protein (AP) complexes are key regulators of cargo sorting into vesicles in yeast and mammals but their existence and function in plants have not been demonstrated. Here we report the identification of the protein-affected trafficking 4 (pat4) mutant defective in the putative δ subunit of the AP-3 complex. pat4 and pat2, a mutant isolated from the same GFP imaging-based forward genetic screen that lacks a functional putative AP-3 β, as well as dominant negative AP-3 μ transgenic lines display undistinguishable phenotypes characterized by largely normal morphology and development, but strong intracellular accumulation of membrane proteins in aberrant vacuolar structures. All mutants are defective in morphology and function of lytic and protein storage vacuoles (PSVs) but show normal sorting of reserve proteins to PSVs. Immunoprecipitation experiments and genetic studies revealed tight functional and physical associations of putative AP-3 β and AP-3 δ subunits. Furthermore, both proteins are closely linked with putative AP-3 μ and σ subunits and several components of the clathrin and dynamin machineries. Taken together, these results demonstrate that AP complexes, similar to those in other eukaryotes, exist in plants, and that AP-3 plays a specific role in the regulation of biogenesis and function of vacuoles in plant cells.     

The AtRAD51C gene is required for normal meiotic chromosome synapsis and double-stranded break repair in Arabidopsis

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.     

Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system

Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase(+)/nuclease(-) and ATPase(-)/nuclease(+)) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade-crRNA complex targeting foreign DNA.     

ALS3 encodes a phloem-localized ABC transporter-like protein that is required for aluminum tolerance in Arabidopsis

Aluminum (Al) toxicity in acid soils is a worldwide agricultural problem that severely limits crop productivity through inhibition of root growth. Previously, Arabidopsis mutants with increased Al sensitivity were isolated in order to identify genes important for Al tolerance in plants. One mutant, als3, exhibited extreme root growth inhibition in the presence of Al, suggesting that this mutation negatively impacts a gene required for Al tolerance. Map-based cloning of the als3-1 mutation resulted in the isolation of a novel gene that encodes a previously undescribed ABC transporter-like protein, which is highly homologous to a putative bacterial metal resistance protein, ybbM. Northern analysis for ALS3 expression revealed that it is found in all organs examined, which is consistent with the global nature of Al sensitivity displayed by als3, and that expression increases in roots following Al treatment. Based on GUS fusion and in situ hybridization analyses, ALS3 is primarily expressed in leaf hydathodes and the phloem throughout the plant, along with the root cortex following Al treatment. Immunolocalization indicates that ALS3 predominantly accumulates in the plasma membrane of cells that express ALS3. From our results, it appears that ALS3 encodes an ABC transporter-like protein that is required for Al resistance/tolerance and may function to redistribute accumulated Al away from sensitive tissues in order to protect the growing root from the toxic effects of Al.     

The Arabidopsis UVH1 gene is a homolog of the yeast repair endonuclease RAD1

Ultraviolet radiation induces DNA damage products, largely in the form of pyrimidine dimers, that are both toxic and mutagenic. In most organisms, including Arabidopsis, these lesions are repaired both through a dimer-specific photoreactivation mechanism and through a less efficient light-independent mechanism. Several mutants defective in this "dark repair" pathway have been previously described. The mechanism of this repair has not been elucidated, but is thought to be homologous to the nucleotide excision repair mechanisms found in other eukaryotes. Here we report the complementation of the Arabidopsis uvh1 dark repair mutant with the Arabidopsis homolog of the yeast nucleotide excision repair gene RAD1, which encodes one of the subunits of the 5'-repair endonuclease. The uvh1-2 mutant allele carries a glycine-->aspartate amino acid change that has been previously identified to produce a null allele of RAD1 in yeast. Although Arabidopsis homologs of genes involved in nucleotide excision repair are readily identified by searching the genomic database, it has not been established that these homologs are actually required for dark repair in plants. The complementation of the Arabidopsis uvh1 mutation with the Arabidopsis RAD1 homolog clearly demonstrates that the mechanism of nucleotide excision repair is conserved among the plant, animal, and fungal kingdoms.     

The MER3 helicase involved in meiotic crossing over is stimulated by single-stranded DNA-binding proteins and unwinds DNA in the 3' to 5' direction

The meiosis-specific MER3 protein of Saccharomyces cerevisiae is required for crossing over, which ensures faithful segregation of homologous chromosomes at the first meiotic division. The predicted sequence of the MER3 protein contains the seven motifs characteristic of the DExH-box type of DNA/RNA helicases. The purified MER3 protein is a DNA helicase, which can displace a 50-nucleotide fragment annealed to a single-stranded circular DNA. MER3 was found to have ATPase activity, which was stimulated either by single- or double-stranded DNA. The turnover rate, k(cat), of ATP hydrolysis was approximately 500/min in the presence of either DNA. MER3 was able to efficiently displace relatively long 631-nucleotide fragments from single-stranded circular DNA only in the presence of the S. cerevisiae single-stranded DNA-binding protein, RPA (replication protein A). It appears that RPA inhibits re-annealing of the single-stranded products of the MER3 helicase. The MER3 helicase was found to unwind DNA in the 3' to 5' direction relative to single-stranded regions in the DNA substrates. Possible roles for the MER3 helicase in meiotic crossing over are discussed.     

The Drosophila embargoed gene is required for larval progression and encodes the functional homolog of schizosaccharomyces Crm1

The CRM1 (Exportin 1) protein is a receptor for leucine-rich nuclear export signal sequences. We have molecularly characterized the Drosophila melanogaster embargoed (emb) gene and find that it encodes a product with 49 and 71% sequence identity to the fission yeast Schizosaccharomyces pombe and human CRM1 proteins, respectively. We show that expression of the emb cDNA is sufficient to suppress the growth phenotype of both conditional-lethal and null S. pombe crm1(-) mutant strains, suggesting that emb encodes the functional homologue of the S. pombe Crm1 protein. Through mutagenesis screens we have recovered a series of recessive lethal emb mutations. There is a substantial maternal contribution of emb mRNA and animals hemizygous for our emb alleles can develop to second instar larvae but persist at this stage and consistently fail to undergo the molt to the third instar stage. We see a nuclear accumulation of endogenous actin in the intestinal epithelial cells of the emb mutant larvae, consistent with a role for the emb gene product in nuclear export of actin protein.     

The Arabidopsis SOS5 locus encodes a putative cell surface adhesion protein and is required for normal cell expansion

Cell surface proteoglycans have been implicated in many aspects of plant growth and development, but genetic evidence supporting their function has been lacking. Here, we report that the Salt Overly Sensitive5 (SOS5) gene encodes a putative cell surface adhesion protein and is required for normal cell expansion. The sos5 mutant was isolated in a screen for Arabidopsis salt-hypersensitive mutants. Under salt stress, the root tips of sos5 mutant plants swell and root growth is arrested. The root-swelling phenotype is caused by abnormal expansion of epidermal, cortical, and endodermal cells. The SOS5 gene was isolated through map-based cloning. The predicted SOS5 protein contains an N-terminal signal sequence for plasma membrane localization, two arabinogalactan protein-like domains, two fasciclin-like domains, and a C-terminal glycosylphosphatidylinositol lipid anchor signal sequence. The presence of fasciclin-like domains, which typically are found in animal cell adhesion proteins, suggests a role for SOS5 in cell-to-cell adhesion in plants. The SOS5 protein was present at the outer surface of the plasma membrane. The cell walls are thinner in the sos5 mutant, and those between neighboring epidermal and cortical cells in sos5 roots appear less organized. SOS5 is expressed ubiquitously in all plant organs and tissues, including guard cells in the leaf.     

The UCU1 Arabidopsis gene encodes a SHAGGY/GSK3-like kinase required for cell expansion along the proximodistal axis

Most signal transduction pathways central to development are not shared by plants and animals. Such is the case of the Wingless/Wnt signaling pathway, whose components play key roles in metazoan pattern formation and tumorigenesis, but are absent in plants, with the exception of SHAGGY/GSK3, a cytoplasmic protein kinase represented in the genome of Arabidopsis thaliana by a family of 10 AtSK genes for which mutational evidence is scarce. Here, we describe the characterization of mutant alleles of the Arabidopsis ULTRACURVATA1 (UCU1) gene, the two strongest of which dramatically reduce cell expansion along the proximodistal axis, dwarfing the mutant plants, whose cells expand properly across but not along most organs. Proximodistal expansion of adaxial (dorsal) and abaxial (ventral) leaf cells exhibits a differential dependence on UCU1 function, as suggested by the leaves of ucu1 mutants, which are rolled spirally downward in a circinate manner. We have positionally cloned the UCU1 gene, which encodes an AtSK protein involved in the cross-talk between auxin and brassinosteroid signaling pathways, as indicated by the responses of ucu1 mutants to plant hormones and the phenotypes of double mutants involving ucu1 alleles.     

The END3 gene encodes a protein that is required for the internalization step of endocytosis and for actin cytoskeleton organization in yeast.

Two Saccharomyces cerevisiae mutants, end3 and end4, defective in the internalization step of endocytosis, have previously been isolated. The END3 gene was cloned by complementation of the temperature-sensitive growth defect caused by the end3 mutation and the END3 nucleotide sequence was determined. The END3 gene product is a 40-kDa protein that has a putative EF-hand Ca(2+)-binding site, a consensus sequence for the binding of phosphotidylinositol 4,5-bisphosphate (PIP2), and a C-terminal domain containing two homologous regions of 17-19 aa. The EF-hand consensus and the putative PIP2-binding sites are seemingly not required for End3 protein function. In contrast, different portions of the End3p N-terminal domain, and at least one of the two repeated regions in its C-terminus, are required for End3p activity. Disruption of the END3 gene yielded cells with the same phenotype as the original end3 mutant. An end3ts allele was obtained and this allowed us to demonstrate that End3p is specifically involved in the internalization step of endocytosis. In addition, End3p was shown to be required for proper organization of the actin cytoskeleton and for the correct distribution of chitin at the cell surface.