The matrix (M) protein of vesicular stomatitis virus (VSV) is a potent inhibitor of bidirectional nuclear transport. Here we demonstrate that inhibition occurs when M protein is in the nucleus of Xenopus laevis oocytes and that M activity is readily reversed by a monoclonal antibody (␣M). We identify a region of M protein, amino acids 51 to 59, that is required both for inhibition of transport and for efficient recognition by ␣M. When expressed in transfected HeLa cells, M protein colocalizes with nuclear pore complexes (NPCs) at the nuclear rim. Moreover, mutation of a single amino acid, methionine 51, eliminates both transport inhibition and targeting to NPCs. We propose that M protein inhibits bidirectional transport by interacting with a component of the NPC or an NPC-associated factor that participates in nucleocytoplasmic transport.Trafficking of large macromolecules (more than 50 kDa) between the nucleus and the cytoplasm occurs through nuclear pore complexes (NPCs) via signal-dependent, carrier-mediated processes (reviewed in references 39 and 54). This transport is subject to control in response to a variety of stimuli such as progression through the cell cycle, exposure to stress, and infection by viruses (reviewed in reference 39). Thus, control of nucleocytoplasmic transport is an important element in the regulation of gene expression.Much of the carrier-mediated movement through NPCs requires cargo-specific transport receptors called importins and exportins (or karyopherins), which are members of the importin ␤ superfamily of proteins (16,21,45,61). Transport receptors can bind their cargoes either directly or via specialized adapter proteins. For example, importin ␤ mediates import of proteins containing basic nuclear localization sequences and small nuclear ribonucleoproteins (snRNPs) using the adapter proteins importin ␣ (1) and snurportin (28, 43), respectively. Importin ␤ can also interact directly with import cargoes, such as cyclin B (40, 53) and certain ribosomal proteins (30). CRM1 (Exportin1) mediates the export of proteins containing leucine-rich nuclear export signals (NES) as well as unspliced viral mRNAs and pre-snRNAs that are bound to specific NEScontaining adapter proteins (14,17,51,57). Exportin-t binds directly to its RNA export cargo, tRNA (3, 33).Directionality of nuclear transport appears to be governed largely by Ran, a small GTPase that is a central component of most known nucleocytoplasmic transport pathways (reviewed in references 9 and 41). Owing to the asymmetric localization of the Ran effector proteins RanGAP (the GTPase activating protein in the cytoplasm) and RCC1 (the guanine nucleotide exchange factor in the nucleus), a steep concentration gradient of RanGTP is presumed to exist across the nuclear envelope (29). This gradient plays a pivotal role in nucleocytoplasmic transport by triggering both assembly and disassembly of receptor-cargo complexes in the appropriate subcellular compartment (60). Thus, import complexes assemble in the cytoplasm in the absence of RanG...

Many eubacterial DNA polymerases are bifunctional molecules having both polymerization (P) and 5 nuclease (N) activities, which are contained in separable domains. We previously showed that the DNA polymerase I of Thermus aquaticus (TaqNP) endonucleolytically cleaves DNA substrates, releasing unpaired 5 arms of bifurcated duplexes. Here, we compare the substrate specificities of TaqNP and the isolated 5 nuclease domain of this enzyme, TaqN. Both enzymes are significantly activated by primer oligonucleotides that are hybridized to the 3 arm of the bifurcation; optimal stimulation requires overlap of the 3 terminal nucleotide of the primer with the terminal base pair of the duplex, but the terminal nucleotide need not hybridize to the complementary strand in the substrate. In the presence of Mn 2؉ ions, TaqN can cleave both RNA and circular DNA at structural bifurcations. Certain anti-TaqNP mAbs block cleavage by one or both enzymes, whereas others can stimulate cleavage of nonoptimal substrates.Structure-sensing nucleases are ubiquitous in biology, being essential for both the synthesis and the repair of DNA (1-12). Several of these enzymes cleave bifurcated duplex DNAs endonucleolytically, releasing the single-stranded 5Ј arm (13). The 5Ј nuclease activity accounts for the ability of these enzymes to remove RNA primers or damaged DNA nucleotides (for review, see ref. 15). In eubacteria, 5Ј nucleases are discrete domains in the DNA polymerases, but in Eukarya and Archaea, they are separate from DNA polymerases and have been called DNA endonuclease IV (1) or, more recently, FEN1 nuclease (16). We refer to the DNA polymerase of T. aquaticus as TaqNP, because it contains both the nuclease and polymerase domains in a single polypeptide; likewise, we refer to the isolated nuclease domain of this enzyme as TaqN.We previously showed that the 5Ј nuclease activity of TaqNP is increased by several orders of magnitude if an oligonucleotide (the primer) is hybridized to the 3Ј arm of the bifurcation (13). The role of the primer in the activation and location of cleavage was unclear. We also showed that cleavage required a free 5Ј end of the single-stranded arm, indicating that the enzyme moved to the site of cleavage by threading the single strand through a hole or a narrow groove in the enzyme, and a requirement for a free 5Ј end was subsequently observed for the calf FEN1 nuclease (14). Recent crystal structures have demonstrated the existence of helical arches or holes in a similar nuclease, T5 exonuclease, as well as in .The influence of the polymerase domain on the activity of the 5Ј nuclease has not been determined. Structure-function probing of the nuclease and polymerase domains of TaqNP by using mAbs generated against the intact enzyme (22) demonstrated some functional overlap between the nuclease and polymerase domains of the enzyme (23). Here, we compare the activities and substrate requirements of TaqN, the isolated 5Ј nuclease of TaqNP, and the same functional domain when it is part of the intact TaqNP h...

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The mounting and critical need for scientific data curation professionals was the impetus for the Data Curation Education in Research Centers (DCERC) program. DCERC is developing a sustainable and transferable model for educating Library and Information Science (LIS) students in data curation through field experiences in research and data centers. DCERC has established and implemented a graduate research and education program bringing students into the real world of scientific data curation, where they engage with current practices and challenges, and share their developing expertise and research. The DCERC partner institutions are developing and evaluating this model with the intention of scaling the program to a larger cadre of partners and participants. This paper reports on progress in the early phases of the model development.

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