GEN1/Yen1 and the SLX4 complex: solutions to the problem of Holliday junction resolution

Chromosomal double-strand breaks (DSBs) are considered to be among the most deleterious DNA lesions found in eukaryotic cells due to their propensity to promote genome instability. DSBs occur as a result of exogenous or endogenous DNA damage, and also occur during meiotic recombination. DSBs are often repaired through a process called homologous recombination (HR), which employs the sister chromatid in mitotic cells or the homologous chromosome in meiotic cells, as a template for repair. HR frequently involves the formation and resolution of four-way DNA structures referred to as the Holliday junction (HJ). Despite extensive study, the machinery and mechanisms used to process these structures in eukaryotes have remained poorly understood. Recent work has identified XPG and UvrC/GIY domain-containing structure-specific endonucleases that can symmetrically cleave HJs in vitro in a manner that allows for religation without additional processing, properties that are reminiscent of the classical RuvC HJ resolvase in bacteria. Genetic studies reveal potential roles for these HJ resolvases in repair after DNA damage and during meiosis. The stage is now set for a more comprehensive understanding of the specific roles these enzymes play in the response of cells to DSBs, collapsed replication forks, telomere dysfunction, and meiotic recombination.     

Roles of SGS1, MUS81, and RAD51 in the repair of lagging-strand replication defects in Saccharomyces cerevisiae

Yeast cells lacking the SGS1 DNA helicase and the MUS81 structure-specific endonuclease display a synthetic lethality that is suppressed by loss of the RAD51 recombinase. This epistatic interaction suggests that the primary function of SGS1 or MUS81, or both genes, is downstream of RAD51. To identify RAD51-independent functions of SGS1 and MUS81, a synthetic-lethal screen was performed on the sgs1 mus81 rad51triple mutant. We found that mutation of RNH202, which encodes a subunit of the hetero-trimeric RNase H2, generates a profound synthetic-sickness in this background. RNase H2 is thought to play a non-essential role in Okazaki fragment maturation. Cells lacking RNH202 showed synthetic growth defects when combined with either mus81 or sgs1 alone. But, whereas the loss of RAD51 had little effect on rnh202 sgs1 double mutants, it strongly inhibited the growth of rnh202 mus81 cells. These data indicate that the primary function of SGS1, but not MUS81, is downstream of RAD51. SGS1 must have some RAD51-independent function, however, since the growth of rnh202 mus81 rad51cells was further compromised by the loss of SGS1. Consistent with these results, we show that rnh202 cells display a sensitivity to DNA-damaging agents that is exacerbated in the absence of RAD51 or MUS81. These data support a model in which defects in lagging-strand replication are repaired by the Mus81 endonuclease or through a pathway dependent on Rad51 and Sgs1.     

More complexity to the Bloom's syndrome complex

Bloom's syndrome is caused by mutations in the BLM gene. The BLM gene product, BLM helicase, forms a complex with two other proteins, DNA topoisomerase IIIalpha and RMI1. In this issue of Genes & Development, Wang and colleagues (2843-2855) and Meetei and colleagues (2856-2868) report the discovery of a fourth component of this complex called RMI2. RMI2 may be a representative of a new family of OB-fold-containing proteins that are important for complex stabilization and checkpoint response.     

Crystal structure of the Gtr1p-Gtr2p complex reveals new insights into the amino acid-induced TORC1 activation

The target of rapamycin (TOR) complex 1 (TORC1) is a central cell growth regulator in response to a wide array of signals. The Rag GTPases play an essential role in relaying amino acid signals to TORC1 activation through direct interaction with raptor and recruitment of the TORC1 complex to lysosomes. Here we present the crystal structure of the Gtr1p–Gtr2p complex, the Rag homologs from Saccharomyces cerevisiae, at 2.8 Å resolution. The heterodimeric GTPases reveal a pseudo-twofold symmetric organization. Structure-guided functional analyses of RagA–RagC, the human homologs of Gtr1p–Gtr2p, show that both G domains (N-terminal GTPase domains) and dimerization are important for raptor binding. In particular, the switch regions of the G domain in RagA are indispensible for interaction with raptor, and hence TORC1 activation. The dimerized C-terminal domains of RagA–RagC display a remarkable structural similarity to MP1/p14, which is in a complex with lysosome membrane protein p18, and directly interact with p18, therefore recruiting mTORC1 to the lysosome for activation by Rheb. Our results reveal a structural model for the mechanism of the Rag GTPases in TORC1 activation and amino acid signaling.     

Phosphorylation-dependent regulation of the F-BAR protein Hof1 during cytokinesis

Spatial and timely coordination of cytokinesis is crucial for the maintenance of organelle inheritance and genome integrity. The mitotic exit network (MEN) pathway controls both the timely initiation of mitotic exit and cytokinesis in budding yeast. Here we identified the conserved F-BAR protein Hof1 as a substrate of the MEN kinase complex Dbf2-Mob1 during cytokinesis. We show that polo-like kinase Cdc5 first phosphorylates Hof1 to allow subsequent phosphorylation by Dbf2-Mob1. This releases Hof1 from the septin ring and facilitates Hof1 binding to the medial actomyosin ring (AMR), where Hof1 promotes AMR contraction and membrane ingression. Domain structure analysis established that the central, unstructured, region of Hof1, named the ring localization sequence (RLS), is sufficient to mediate Hof1's binding to the medial ring in a cell cycle-dependent manner. Genetic and functional data support a model in which Dbf2-Mob1 regulates Hof1 by inducing domain rearrangements, leading to the exposure of the Hof1 RLS domain during telophase.     

Long-range action of Nodal requires interaction with GDF1

GDF1 (growth/differentiation factor 1), a Vg1-related member of the transforming growth factor-beta superfamily, is required for left-right patterning in the mouse, but the precise function of GDF1 has remained largely unknown. In contrast to previous observations, we now show that GDF1 itself is not an effective ligand but rather functions as a coligand for Nodal. GDF1 directly interacts with Nodal and thereby greatly increases its specific activity. Gdf1 expression in the node was found necessary and sufficient for initiation of asymmetric Nodal expression in the lateral plate of mouse embryos. Coexpression of GDF1 with Nodal in frog embryos increased the range of the Nodal signal. Introduction of Nodal alone into the lateral plate of Gdf1 knockout mouse embryos did not induce Lefty1 expression at the midline, whereas introduction of both Nodal and GDF1 did, showing that GDF1 is required for long-range Nodal signaling from the lateral plate to the midline. These results suggest that GDF1 regulates the activity and signaling range of Nodal through direct interaction.     

Cdc7-Drf1 kinase links chromosome cohesion to the initiation of DNA replication in Xenopus egg extracts

To establish functional cohesion between replicated sister chromatids, cohesin is recruited to chromatin before S phase. Cohesin is loaded onto chromosomes in the G1 phase by the Scc2-Scc4 complex, but little is known about how Scc2-Scc4 itself is recruited to chromatin. Using Xenopus egg extracts as a vertebrate model system, we showed previously that the chromatin association of Scc2 and cohesin is dependent on the prior establishment of prereplication complexes (pre-RCs) at origins of replication. Here, we report that Scc2-Scc4 exists in a stable complex with the Cdc7-Drf1 protein kinase (DDK), which is known to bind pre-RCs and activate them for DNA replication. Immunodepletion of DDK from Xenopus egg extracts impairs chromatin association of Scc2-Scc4, a defect that is reversed by wild-type, but not catalytically inactive DDK. A complex of Scc4 and the N terminus of Scc2 is sufficient for chromatin loading of Scc2-Scc4, but not for cohesin recruitment. These results show that DDK is required to tether Scc2-Scc4 to pre-RCs, and they underscore the intimate link between early steps in DNA replication and cohesion.     

PX-RICS mediates ER-to-Golgi transport of the N-cadherin/beta-catenin complex

Cadherins mediate Ca2+-dependent cell-cell adhesion. Efficient export of cadherins from the endoplasmic reticulum (ER) is known to require complex formation with beta-catenin. However, the molecular mechanisms underlying this requirement remain elusive. Here we show that PX-RICS, a beta-catenin-interacting GTPase-activating protein (GAP) for Cdc42, mediates ER-to-Golgi transport of the N-cadherin/beta-catenin complex. Knockdown of PX-RICS expression induced the accumulation of the N-cadherin/beta-catenin complex in the ER and ER exit site, resulting in a decrease in cell-cell adhesion. PX-RICS was also required for ER-to-Golgi transport of the fibroblast growth factor-receptor 4 (FGFR4) associated with N-cadherin. PX-RICS-mediated ER-to-Golgi transport was dependent on its interaction with beta-catenin, phosphatidylinositol-4-phosphate (PI4P), Cdc42, and its novel binding partner gamma-aminobutyric acid type A receptor-associated protein (GABARAP). These results suggest that PX-RICS ensures the efficient entry of the N-cadherin/beta-catenin complex into the secretory pathway, and thereby regulates the amount of N-cadherin available for cell adhesion and FGFR4-mediated signaling.     

Reconstitution and structural analysis of the yeast box H/ACA RNA-guided pseudouridine synthase

Box H/ACA ribonucleoprotein particles (RNPs) mediate pseudouridine synthesis, ribosome formation, and telomere maintenance. The structure of eukaryotic H/ACA RNPs remains poorly understood. We reconstituted functional Saccharomyces cerevisiae H/ACA RNPs with recombinant proteins Cbf5, Nop10, Gar1, and Nhp2 and a two-hairpin H/ACA RNA; determined the crystal structure of a Cbf5, Nop10, and Gar1 ternary complex at 1.9 Å resolution; and analyzed the structure–function relationship of the yeast complex. Although eukaryotic H/ACA RNAs have a conserved two-hairpin structure, isolated single-hairpin RNAs are also active in guiding pseudouridylation. Nhp2, unlike its archaeal counterpart, is largely dispensable for the activity, reflecting a functional adaptation of eukaryotic H/ACA RNPs to the variable RNA structure that Nhp2 binds. The N-terminal extension of Cbf5, a hot spot for dyskeratosis congenita mutation, forms an extra structural layer on the PUA domain. Gar1 is distinguished from the assembly factor Naf1 by containing a C-terminal extension that controls substrate turnover and the Gar1–Naf1 exchange during H/ACA RNP maturation. Our results reveal significant novel features of eukaryotic H/ACA RNPs.     

Crystal structure and putative substrate identification for the Entamoeba histolytica low molecular weight tyrosine phosphatase

Entamoeba histolytica is a eukaryotic intestinal parasite of humans, and is endemic in developing countries. We have characterized the E. histolytica putative low molecular weight protein tyrosine phosphatase (LMW-PTP). The structure for this amebic tyrosine phosphatase was solved, showing the ligand-induced conformational changes necessary for binding of substrate. In amebae, it was expressed at low but detectable levels as detected by immunoprecipitation followed by immunoblotting. A mutant LMW-PTP protein in which the catalytic cysteine in the active site was replaced with a serine lacked phosphatase activity, and was used to identify a number of trapped putative substrate proteins via mass spectrometry analysis. Seven of these putative substrate protein genes were cloned with an epitope tag and overexpressed in amebae. Five of these seven putative substrate proteins were demonstrated to interact specifically with the mutant LMW-PTP. This is the first biochemical study of a small tyrosine phosphatase in Entamoeba, and sets the stage for understanding its role in amebic biology and pathogenesis.     

Role of IL-18 in the secretion of Il-1beta, sIL-1RII, and IL-1Ra by human neutrophils

In the present study we investigated the effect of IL-18 on the production of IL-1β, IL-1Ra and sIL-1RII by human neutrophils. Our observations indicate that rhIL-18 induces IL-1β and, to a lesser extend, IL-1Ra and sIL-1RII production by human neutrophils isolated form peripheral blood. However, this effect was less important in comparison with LPS-stimulation. Moreover, the results obtained suggest that IL-18 can induce priming of neutrophils for IL-1β and, to a lesser extend, IL-1Ra and sIL-1RII production by LPS-stimulated cells. The capacity of IL-18 to serve as an effective modulator for IL-1β and its regulatory proteins may have significance in the inflammatory and immune reactions mediated by IL-1β.     

Ligation of TAPA-1 (CD81) or major histocompatibility complex class II in co-cultures of human B and T lymphocytes enhances interleukin-4 synthesis by antigen-specific CD4+ T cells

We have previously shown that CD4⁺ T cells from allergic individuals are predisposed to producing interleukin (IL)-4 in response to allergens. IL-4 production could be modulated by antigen concentration as well as by the type of antigen-presenting cells (APC), with B lymphocytes inducing greater quantities of IL-4 than monocytes. Using this system we examined IL-4 synthesis after culture of CD4⁺ T cells with B cells, monocytes, or both, as APC in the presence of allergen and a monoclonal antibody against CD81 (TAPA-1), a member of the TM4 superfamily of proteins that regulates activation, proliferation and trafficking of B cells. Addition of anti-CD81 mAb during culture enhanced IL-4 synthesis by 2- to 70-fold over that using an isotype-matched control mAb. Furthermore, anti-CD81 mAb enhanced IL-4 synthesis in CD4⁺ T cells only when CD4⁺ T cells were cultured with B cells but not monocytes as APC, indicating that anti-CD81 mAb affected IL-4 synthesis in T cells via interactions with B cells. However, pretreatment of either population separately with anti-CD81 mAb prior to culture had no effect on subsequent IL-4 synthesis, suggesting a requirement for temporal or cooperative interactions between T and B lymphocytes. In addition, anti-CD81 mAb enhanced IL-4 production but reduced CD4⁺ T cell antigen-specific proliferation, demonstrating that IL-4 production and proliferation by CD4⁺ T cells were inversely related. Finally, mAb to major histocompatibility complex class II but not to anti-CD19 also enhanced IL-4 synthesis when B lymphocytes were used as APC. In all instances, enhancement of CD4⁺ IL-4 synthesis correlated with the presence of large cell aggregates in T-B lymphocyte cocultures. These results indicate that the capacity of B cells to induce IL-4 can be significantly enhanced by ligation of particular molecules on their surface and should aid in the design of treatments for diseases in which modulation of the cytokine profile would be beneficial.     

A Korean female patient with thiamine-responsive pyruvate dehydrogenase complex deficiency due to a novel point mutation (Y161C)in the PDHA1 gene

Pyruvate dehydrogenase complex (PDHC) deficiency is mostly due to mutations in the X-linked E1alpha subunit gene (PDHA1). Some of the patients with PDHC deficiency showed clinical improvements with thiamine treatment. We report the results of biochemical and molecular analysis in a female patient with lactic acidemia. The PDHC activity was assayed at different concentrations of thiamine pyrophosphate (TPP). The PDHC activity showed null activity at low TPP concentration (1 x 10(-3) mM), but significantly increased at a high TPP concentration (1 mM). Sequencing analysis of PDHA1 gene of the patient revealed a substitution of cysteine for tyrosine at position 161 (Y161C). Thiamine treatment resulted in reduction of the patient's serum lactate concentration and dramatic clinical improvement. Biochemical, molecular, and clinical data suggest that this patient has a thiamine-responsive PDHC deficiency due to a novel mutation, Y161C. Therefore, to detect the thiamine responsiveness it is necessary to measure activities of PDHC not only at high but also at low concentration of TPP.     

Generation of human neutralizing monoclonal antibodies against the 2009 pandemic H1N1 virus from peripheral blood memory B lymphocytes

The 2009 H1N1 influenza pandemic demonstrated the significance of a global health threat to human beings. Although pandemic H1N1 vaccines have been rapidly developed, passive serotherapy may offer superior immediate protection against infections in children, the elderly and immune-compromised patients during an influenza pandemic. Here, we applied a novel strategy based on Epstein–Barr virus (EBV)-immortalized peripheral blood memory B cells to screen high viral neutralizing monoclonal antibodies (MAbs) from individuals vaccinated with the 2009 pandemic H1N1 vaccine PANFLU.1. Through a massive screen of 13 090 immortalized memory B-cell clones from three selected vaccinees, seven MAbs were identified with both high viral neutralizing capacities and hemagglutination inhibition (HAI) activities against the 2009 pandemic H1N1 viruses. These MAbs may have important clinical implications for passive serotherapy treatments of infected patients with severe respiratory syndrome, especially children, the elderly and immunodeficient individuals. Our successful strategy for generating high-affinity MAbs from EBV-immortalized peripheral blood memory B cells may also be applicable to other infectious or autoimmune diseases.