Replication protein A large subunit (RPA1a) limits chiasma formation during rice meiosis

Replication protein A (RPA), a single-stranded DNA-binding protein, plays essential role in homologous recombination. However, because deletion of RPA causes embryonic lethality in mammals, the exact function of RPA in meiosis remains unclear. In this study, we generated an rpa1a mutant using CRISPR/Cas9 technology and explored its function in rice (Oryza sativa) meiosis. In rpa1a, 12 bivalents were formed at metaphase I, just like in wild-type, but chromosome fragmentations were consistently observed at anaphase I. Fluorescence in situ hybridization assays indicated that these fragmentations were due to the failure of the recombination intermediates to resolve. Importantly, the mutant had a highly elevated chiasma number, and loss of RPA1a could completely restore the 12 bivalent formations in the zmm (for ZIP1-4, MSH4/5, and MER3) mutant background. Protein–protein interaction assays showed that RPA1a formed a complex with the methyl methansulfonate and UV sensitive 81 (and the Fanconi anemia complementation group M–Bloom syndrome protein homologs (RECQ4A)–Topoisomerase3α–RecQ-mediated genome instability 1 complex to regulate chiasma formation and processing of the recombination intermediates. Thus, our data establish a pivotal role for RPA1a in promoting the accurate resolution of recombination intermediates and in limiting redundant chiasma formation during rice meiosis.

RPA1a guarantees accurate meiotic recombination during rice gametogenesis, and acts as a guard to prevent excessive meiotic crossovers.     

Localisation of phosphorylated MAP kinase during the transition from meiosis I to meiosis II in pig oocytes

Mitogen-activated protein kinase (MAPK) has been reported to be involved in oocyte maturation in all animals so far examined. In the present study we investigate the expression and localisation of active phosphorylated MAPKs (p44ERK1/p42ERK2) during maturation of pig oocytes. In immunoblot analysis using anti-p44ERK1 antibody which recognised both active and inactive forms of p44ERK1 and p42ERK2, we confirmed that MAPKs were phosphorylated around the time of germinal vesicle breakdown (GVBD) and the active phosphorylated MAPKs (pMAKs) were maintained until metaphase II, as has been reported. On immunofluorescent confocal microscopy using anti-pMAPK antibody which recognised only phosphorylated forms of MAPKs, pMAPK was localised at the spindle poles in pig mitotic cells. On the other hand, in pig oocytes, no signal was detected during GV stage. After GVBD, the area around condensed chromosomes was preferentially stained at metaphase I although whole cytoplasm was faintly stained. At early anaphase I, the polar regions of the meiotic spindle were prominently stained. However, during the progression of anaphase I and telophase I pMAPK was detected at the mid-zone of the elongated spindle, gradually becoming concentrated at the centre. Finally, at the time of emission of the first polar body, pMAPK was detected as a ring-like structure between the condensed chromosomes and the first polar body, and the staining was maintained even after the metaphase II spindle was formed. The inhibition of MAPK activity with the MAPK kinase inhibitor U0126 during the meiosis I/meiosis II transition suppressed chromosome separation, first polar body emission and formation of the metaphase II spindle. From these results, we propose that the spindle-associated pMAPKs play an important role in the events occurring during the meiosis I/meiosis II transition, such as chromosome separation, spindle elongation and cleavage furrow formation in pig oocytes.     

The retinoblastoma protein is phosphorylated during specific phases of the cell cycle

p105-RB is the product of the retinoblastoma tumor suppressor gene. It is a nuclear phosphoprotein hypothesized to act as an inhibitor of cellular proliferation, yet surprisingly it is present in actively dividing cells. To look for changes in p105-RB that may regulate its activity during the cell cycle, we generated synchronized cell populations and followed their progression through the cell cycle. p105-RB is synthesized throughout the cycle, but is phosphorylated in a phase-specific manner. In the G0 and G1 phases of the cell cycle, an unphosphorylated species of the protein is the only detectable form, whereas in the S and G2/M phases, multiple phosphorylated species of p105-RB are detected.     

Ecm11 protein of yeast Saccharomyces cerevisiae is regulated by sumoylation during meiosis

Damaged regulation of the small ubiquitin-like modifier (SUMO) system contributes to some human diseases; therefore, it is very important to identify the SUMO targets and to determine the function of their sumoylation. In this study, it is shown that Ecm11 protein in Saccharomyces cerevisiae is modified by SUMO during meiosis. It is known that Ecm11 is required in the early stages of yeast meiosis where its function is related to DNA replication and crossing over. Here it is shown that the level of Ecm11 protein is low in mitosis, but high in meiosis. The highest level of Ecm11 is in the early-middle phase of sporulation. A specific site of sumoylation was identified in Ecm11 at Lys5 and evidence is provided that sumoylation at this site directly regulates Ecm11 function in meiosis. On the other hand, no relationship was observed between sumoylation of Ecm11 and its role during vegetative growth. It was shown that Ecm11 interacts with Siz2 SUMO ligase in a two-hybrid system; although Siz2 is not essential for the Ecm11 sumoylation.     

Nuclear compartmentalization is abolished during fission yeast meiosis

In eukaryotic cells, the nuclear envelope partitions the nucleus from the cytoplasm. The fission yeast Schizosaccharomyces pombe undergoes closed mitosis in which the nuclear envelope persists rather than being broken down, as in higher eukaryotic cells. It is therefore assumed that nucleocytoplasmic transport continues during the cell cycle. Here we show that nuclear transport is, in fact, abolished specifically during anaphase of the second meiotic nuclear division. During that time, both nucleoplasmic and cytoplasmic proteins disperse throughout the cell, reminiscent of the open mitosis of higher eukaryotes, but the architecture of the S. pombe nuclear envelope itself persists. This functional alteration of the nucleocytoplasmic barrier is likely induced by spore wall formation, because ectopic induction of sporulation signaling leads to premature dispersion of nucleoplasmic proteins. A photobleaching assay demonstrated that nuclear envelope permeability increases abruptly at the onset of anaphase of the second meiotic division. The permeability was not altered when sporulation was inhibited by blocking the trafficking of forespore-membrane vesicles from the endoplasmic reticulum to the Golgi. The evidence indicates that yeast gametogenesis produces vesicle transport-mediated forespore membranes by inducing nuclear envelope permeabilization.     

The microtubule-associated protein tau is phosphorylated by Syk

Aberrant phosphorylation of tau protein on serine and threonine residues has been shown to be critical in neurodegenerative disorders called tauopathies. An increasing amount of data suggest that tyrosine phosphorylation of tau might play an equally important role in pathology, with at least three putative tyrosine kinases of tau identified to date. It was recently shown that the tyrosine kinase Syk could efficiently phosphorylate alpha-synuclein, the aggregated protein found in Parkinson's disease and other synucleinopathies. We report herein that Syk is also a tau kinase, phosphorylating tau in vitro and in CHO cells when both proteins are expressed exogenously. In CHO cells, we have also demonstrated by co-immunoprecipitation that Syk binds to tau. Finally, by site-directed mutagenesis substituting the tyrosine residues of tau with phenylalanine, we established that tyrosine 18 was the primary residue in tau phosphorylated by Syk. The identification of Syk as a common tyrosine kinase of both tau and alpha-synuclein may be of potential significance in neurodegenerative disorders and also in neuronal physiology. These results bring another clue to the intriguing overlaps between tauopathies and synucleinopathies and provide new insights into the role of Syk in neuronal physiology.     

Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-stranded breaks (DSBs). These DSBs undergo a 5' --> 3' resection to produce 3' single-stranded DNA ends that serve to channel DSBs into the RAD52 recombinational repair pathway. In vitro studies strongly suggest that several proteins of this pathway--Rad51, Rad52, Rad54, Rad55, Rad57, and replication protein A (RPA)--play a role in the strand exchange reaction. Here, we report a study of the meiotic phenotypes conferred by two missense mutations affecting the largest subunit of RPA, which are localized in the protein interaction domain (rfa1-t11) and in the DNA-binding domain (rfa1-t48). We find that both mutant diploids exhibit reduced sporulation efficiency, very poor spore viability, and a 10- to 100-fold decrease in meiotic recombination. Physical analyses indicate that both mutants form normal levels of meiosis-specific DSBs and that the broken ends are processed into 3'-OH single-stranded tails, indicating that the RPA complex present in these rfa1 mutants is functional in the initial steps of meiotic recombination. However, the 5' ends of the broken fragments undergo extensive resection, similar to what is observed in rad51, rad52, rad55, and rad57 mutants, indicating that these RPA mutants are defective in the repair of the Spo11-dependent DSBs that initiate homologous recombination during meiosis.     

Werner syndrome protein is regulated and phosphorylated by DNA-dependent protein kinase

DNA double-strand breaks (DSBs) are a highly mutagenic and potentially lethal damage that occurs in all organisms. Mammalian cells repair DSBs by homologous recombination and non-homologous end joining, the latter requiring DNA-dependent protein kinase (DNA-PK). Werner syndrome is a disorder characterized by genomic instability, aging pathologies and defective WRN, a RecQ-like helicase with exonuclease activity. We show that WRN interacts directly with the catalytic subunit of DNA-PK (DNA-PK(CS)), which inhibits both the helicase and exonuclease activities of WRN. In addition we show that WRN forms a stable complex on DNA with DNA-PK(CS) and the DNA binding subunit Ku. This assembly reverses WRN enzymatic inhibition. Finally, we show that WRN is phosphorylated in vitro by DNA-PK and requires DNA-PK for phosphorylation in vivo, and that cells deficient in WRN are mildly sensitive to ionizing radiation. These data suggest that DNA-PK and WRN may function together in DNA metabolism and implicate WRN function in non-homologous end joining.     

Neurofilament protein is phosphorylated in the squid giant axon

We have observed the phosphorylation of neurofilament protein from squid axoplasm. Phosphorylation is demonstrated by 32P labeling of protein during incubation of axoplasm with [gamma-32P]ATP. When the labeled proteins are separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two bands, at 2.0 x 10(5) daltons and greater than 4 x 10(5) daltons, contain the bulk of the 32P. The 2.0 x 10(5)-dalton phosphorylated polypeptide comigrates on SDS-PAGE with one of the subunits of squid neurofilament protein. Both major phosphorylated polypeptides co-fractionate with neurofilaments in discontinuous sucrose gradient centrifugation and on gel filtration chromatography on Sepharose 4B. The protein-phosphate bond behaves like a phospho-ester, and labeled phospho-serine is identified in an acid hydrolysate of the protein. The generality of this phenomenon in various species and its possible physiological significance are discussed.     

The Drosophila spn-D gene encodes a RAD51C-like protein that is required exclusively during meiosis

In Drosophila, mutations in double-strand DNA break (DSB) repair enzymes, such as spn-B, activate a meiotic checkpoint leading to dorsal-ventral patterning defects in the egg and an abnormal appearance of the oocyte nucleus. Mutations in spn-D cause an array of ovarian phenotypes similar to spn-B. We have cloned the spn-D locus and found that it encodes a protein of 271 amino acids that shows significant homology to the human RAD51C protein. In mammals the spn-B and spn-D homologs, XRCC3 and RAD51C, play a role in genomic stability in somatic cells. To test for a similar role for spn-B and spn-D in double-strand DNA repair in mitotic cells, we analyzed the sensitivity of single and double mutants to DSBs induced by exposure to X rays and MMS. We found that neither singly mutant nor doubly mutant animals were significantly sensitized to MMS or X rays. These results suggest that spn-B and spn-D act in meiotic recombination but not in repair of DSBs in somatic cells. As there is no apparent ortholog of the meiosis-specific DMC1 gene in the Drosophila genome, and given their meiosis-specific requirement, we suggest that spn-B and spn-D may have a function comparable to DMC1.     

The Saccharomyces cerevisiae centromere protein Slk19p is required for two successive divisions during meiosis

Meiotic cell division includes two separate and distinct types of chromosome segregation. In the first segregational event the sister chromatids remain attached at the centromere; in the second the chromatids are separated. The factors that control the order of chromosome segregation during meiosis have not yet been identified but are thought to be confined to the centromere region. We showed that the centromere protein Slk19p is required for the proper execution of meiosis in Saccharomyces cerevisiae. In its absence diploid cells skip meiosis I and execute meiosis II division. Inhibiting recombination does not correct this phenotype. Surprisingly, the initiation of recombination is apparently required for meiosis II division. Thus Slk19p appears to be part of the mechanism by which the centromere controls the order of meiotic divisions.     

Replication protein A in Pyrococcus furiosus is involved in homologous DNA recombination

Single-stranded DNA-binding protein in Bacteria and replication protein A (RPA) in Eukarya play crucial roles in DNA replication, repair, and recombination processes. We identified an RPA complex from the hyperthermophilic archaeon, Pyrococcus furiosus. Unlike the single-peptide RPAs from the methanogenic archaea, Methanococcus jannaschii and Methanothermobacter thermoautotrophicus, P. furiosus RPA (PfuRPA) exists as a stable hetero-oligomeric complex consisting of three subunits, RPA41, RPA14, and RPA32. The amino acid sequence of RPA41 has some similarity to those of the eukaryotic RPA70 subunit and the M. jannaschii RPA. On the other hand, RPA14 and RPA32 do not share homology with any known open reading frames from Bacteria and Eukarya. However, six of eight archaea, whose total genome sequences have been published, have the open reading frame homologous to RPA32. The PfuRPA complex, but not each subunit alone, specifically bound to a single-stranded DNA and clearly enhanced the efficiency of an in vitro strand-exchange reaction by the P. furiosus RadA protein. Moreover, immunoprecipitation analyses showed that PfuRPA interacts with the recombination proteins, RadA and Hjc, as well as replication proteins, DNA polymerases, primase, proliferating cell nuclear antigen, and replication factor C in P. furiosus cells. These results indicate that PfuRPA plays important roles in the homologous DNA recombination in P. furiosus.     

Hepatitis C virus NS5A protein is phosphorylated by casein kinase II

Hepatitis C virus (HCV) has a positive-strand RNA genome that encodes a polyprotein, which is post-translationally processed by cellular and viral proteinases into three structural and six non-structural (NS) proteins. The NS5A protein, expressed in mammalian cells, exists as two phosphorylated forms of 56 kDa and 58 kDa. In this study, we provide evidence for a stable association between NS5A and a protein kinase from rat-1 cells by affinity to immobilized glutathione-S-transferase (GST)-NS5A fusion protein. This protein kinase was associated through the N-terminus of NS5A and was not regulated by cell cycle. The GST-NS5A was also phosphorylated in vitro by the purified casein kinase II (CKII), a member of the CMCG kinase family. Since CKII and the NS5A-associated protein kinase have the same molecular size and property by In-gel kinase assay and an inhibitor treatment test, we conclude that HCV NS5A protein is phosphorylated by CKII.