SUMMARY The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA, at 2.5 Å resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively-charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and non-complementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies.

The deposition of the abundant presynaptic brain protein alpha-synuclein as fibrillary aggregates in neurons or glial cells is a hallmark lesion in a subset of neurodegenerative disorders. These disorders include Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy, collectively referred to as synucleinopathies. Importantly, the identification of missense mutations in the alpha-synuclein gene in some pedigrees of familial PD has strongly implicated alpha-synuclein in the pathogenesis of PD and other synucleinopathies. However, specific post-translational modifications that underlie the aggregation of alpha-synuclein in affected brains have not, as yet, been identified. Here, we show by mass spectrometry analysis and studies with an antibody that specifically recognizes phospho-Ser 129 of alpha-synuclein, that this residue is selectively and extensively phosphorylated in synucleinopathy lesions. Furthermore, phosphorylation of alpha-synuclein at Ser 129 promoted fibril formation in vitro. These results highlight the importance of phosphorylation of filamentous proteins in the pathogenesis of neurodegenerative disorders.

Xeroderma pigmentosum variant (XP-V) is an inherited disorder which is associated with increased incidence of sunlight-induced skin cancers. Unlike other xeroderma pigmentosum cells (belonging to groups XP-A to XP-G), XP-V cells carry out normal nucleotide-excision repair processes but are defective in their replication of ultraviolet-damaged DNA. It has been suspected for some time that the XPV gene encodes a protein that is involved in trans-lesion DNA synthesis, but the gene product has never been isolated. Using an improved cell-free assay for trans-lesion DNA synthesis, we have recently isolated a DNA polymerase from HeLa cells that continues replication on damaged DNA by bypassing ultraviolet-induced thymine dimers in XP-V cell extracts. Here we show that this polymerase is a human homologue of the yeast Rad30 protein, recently identified as DNA polymerase eta. This polymerase and yeast Rad30 are members of a family of damage-bypass replication proteins which comprises the Escherichia coli proteins UmuC and DinB and the yeast Rev1 protein. We found that all XP-V cells examined carry mutations in their DNA polymerase eta gene. Recombinant human DNA polymerase eta corrects the inability of XP-V cell extracts to carry out DNA replication by bypassing thymine dimers on damaged DNA. Together, these results indicate that DNA polymerase eta could be the XPV gene product.

Autotaxin (ATX) is a tumor cell motility–stimulating factor, originally isolated from melanoma cell supernatants. ATX had been proposed to mediate its effects through 5′-nucleotide pyrophosphatase and phosphodiesterase activities. However, the ATX substrate mediating the increase in cellular motility remains to be identified. Here, we demonstrated that lysophospholipase D (lysoPLD) purified from fetal bovine serum, which catalyzes the production of the bioactive phospholipid mediator, lysophosphatidic acid (LPA), from lysophosphatidylcholine (LPC), is identical to ATX. The Km value of ATX for LPC was 25-fold lower than that for the synthetic nucleoside substrate, p-nitrophenyl-tri-monophosphate. LPA mediates multiple biological functions including cytoskeletal reorganization, chemotaxis, and cell growth through activation of specific G protein–coupled receptors. Recombinant ATX, particularly in the presence of LPC, dramatically increased chemotaxis and proliferation of multiple different cell lines. Moreover, we demonstrate that several cancer cell lines release significant amounts of LPC, a substrate for ATX, into the culture medium. The demonstration that ATX and lysoPLD are identical suggests that autocrine or paracrine production of LPA contributes to tumor cell motility, survival, and proliferation. It also provides potential novel targets for therapy of pathophysiological states including cancer.

elicitor ͉ host-pathogen interaction ͉ N-acetylchitooligosaccharides ͉ pathogen-associated molecular patterns H igher plants have the ability to initiate various defense reactions such as hypersensitive responses, production of phytoalexins and antimicrobial proteins, and reinforcement of cell walls when they are infected by various pathogens (1, 2). They can distinguish self and non-self, or detect specific pathogens, through the perception of signal molecules (elicitors) mostly generated͞secreted from pathogens. Fragments of cell surface macromolecules typical of microorganisms such as cell wall polysaccharides, secreted proteins, as well as a flagella protein, often serve as a potent elicitor to induce defense reactions. They are classified as ''general elicitors'' that are commonly found in various microorganisms and induce defense responses in a wide range of plant species. Perception of general elicitors has been thought to play an important role in the basic resistance, or nonhost resistance, of plants to most potential pathogens. It has also been emerged in recent years that the defense systems mediated by the perception of these ''general elicitors'' have a considerable similarity with mammalian innate immunity, in the recognition of pathogen-associated molecular patterns as well as the molecules involved in the perception and transduction of these signal molecules (3).Chitin oligosaccharides (N-acetylchitooligosaccharides) are a representative general elicitor inducing defense responses in a wide range of plant cells including both monocots and dicots (4). Chitin oligosaccharides were reported to induce defense responses also in mammalian and insect cells (4, 5). Interestingly, specific modifications of chitin oligosaccharides by fatty acids, sulfate, or some sugars, generate ''Nod factors'' that induce nodulation in legume roots in the symbiotic interaction with rhizobial bacteria (6). Thus, the recognition of chitin oligosaccharides and related compounds seems to play a fundamental role in the establishment of basal resistance to potential pathogens in plants and in some cases the symbiotic relationships between leguminous plants and rhizobial bacteria.Concerning to the receptor for chitin oligosaccharide elicitor, we previously identified a high-affinity binding protein for this elicitor in the plasma membrane of rice cells by affinity labeling (7). Similar binding proteins were also detected in various plant cells that could respond to the elicitor (8,9). Correlation between the presence of the binding proteins and the elicitor responsiveness of these cells, correlation between the binding specificity and the preference of the structure of chitin oligosaccharides in defense responses, strongly indicated that the binding proteins function as a receptor, or a part of receptor complex, for chitin oligosaccharide elicitor. Here we report the purification of this chitin oligosaccharide elicitor-binding protein (hereafter designated as CEBiP), cloning of the corresponding cDNA and its functional characte...

In plants and animals, small peptide ligands that signal in cell-cell communication have been suggested to be a crucial component of development. A bioassay of single-cell transdifferentation demonstrates that a dodecapeptide with two hydroxyproline residues is the functional product of genes from the CLE family, which includes CLAVATA3 in Arabidopsis. The dodecapeptide suppresses xylem cell development at a concentration of 10(-11) M and promotes cell division. An application, corresponding to all 26 Arabidopsis CLE protein family members, of synthetic dodecapeptides reveals two counteracting signaling pathways involved in stem cell fate.

Tight junctions are cell-cell adhesion structures in epithelial cell sheets that surround organ compartments in multicellular organisms and regulate the permeation of ions through the intercellular space. Claudins are the major constituents of tight junctions and form strands that mediate cell adhesion and function as paracellular barriers. We report the structure of mammalian claudin-15 at a resolution of 2.4 angstroms. The structure reveals a characteristic β-sheet fold comprising two extracellular segments, which is anchored to a transmembrane four-helix bundle by a consensus motif. Our analyses suggest potential paracellular pathways with distinctive charges on the extracellular surface, providing insight into the molecular basis of ion homeostasis across tight junctions.

A number of proteins accumulate in the anaphase spindle midzone, but the interaction and precise role of these proteins in midzone organization remain obscure. Here, we found that the microtubule-bundling protein PRC1 bound separately to the three motor proteins, KIF4, MKLP1 and CENP-E, but not to the chromosomal passenger proteins. In KIF4-deficient cells, the central spindle was disorganized, and all midzone-associated proteins including PRC1 failed to concentrate at the midline, instead being dispersed along the loosened microtubule bundles of the central spindle. This suggests that KIF4 is essential for the organization of central spindles and for midzone formation. In PRC1-deficient cells, no midzone was formed, KIF4 and CENP-E did not localize to the disconnected half-spindle, and MKLP1 and chromosomal passenger proteins localized to discrete subdomains near microtubule plus ends in the half-spindle. Thus, PRC1 is required for interaction of the two half-spindles and for localization of KIF4 and CENP-E. These results suggest that KIF4 and its binding partner PRC1 play essential roles in the organization of central spindles and midzone formation.