No abstract

The product of the Escherichia coli orf17 gene is a novel nucleoside triphosphate pyrophosphohydrolase with a preference for dATP over the other canonical (deoxy)nucleoside triphosphates, and it catalyzes the hydrolysis of dATP through a nucleophilic attack at the ␤-phosphorus to produce dAMP and inorganic pyrophosphate. It has a pH optimum between 8.5 and 9.0, a divalent metal ion requirement with optimal activity at 5 mM Mg 2؉, a K m of 0.8 mM and a k cat of 5.2 s ؊1 at 37°C for dATP. dAMP is a weak competitive inhibitor with a K i of approximately 4 mM, while PP i is a much stronger inhibitor with an apparent K i of approximately 20 M.The enzyme contains the highly conserved signature sequence GXVEX 2 ETX 6 REVXEEX 2 I designating the MutT family of proteins. However, unlike the other nucleoside triphosphate pyrophosphohydrolases with this conserved sequence, the Orf17 protein does not complement the mutT ؊ mutator phenotype, and thus must serve a different biological role in the cell.Members of the MutT family of proteins are categorized by a conserved amino acid motif originally identified as an important functional region in the Escherichia coli MutT and the Streptococcus pneumoniae MutX antimutator proteins (1). This conserved amino acid signature sequence was found, by computer search, to be present in a number of open reading frames broadly distributed throughout nature, from viruses to humans (1, 2), and one of these, orf17 (GenBank D10165, 1992), is the subject of this paper. The orf17 gene is located at 41 min on the E. coli chromosome (3) just upstream of the ruvC gene (4, 5), which codes for a Holliday junction-specific endonuclease (4).Both the MutT and MutX proteins are nucleoside triphosphatases (6, 7), as are the corresponding proteins from Proteus vulgaris (8) and from humans (9), and all four are implicated in preventing mutations. On the other hand, two other proteins containing the MutT signature sequence are not nucleoside triphosphatases. One of them, a nucleoside pyrophosphatase, prefers NADH as its substrate (10), while the other, GDPmannose mannosyl hydrolase, prefers GDP-mannose (11). Neither of these latter two enzymes has been linked to a mutagenic pathway. A feature common to all six enzymes containing the signature sequence is the hydrolysis of a substrate containing a nucleoside diphosphate linkage.In this paper, we describe the cloning and expression of the orf17 gene, and the purification and characterization of the Orf17 protein. Like the first four enzymes described above, it is a nucleoside triphosphatase. However, unlike those four enzymes, which prefer dGTP as a substrate over the other canonical (deoxy)nucleoside triphosphates, Orf17 has a unique preference for dATP, and it does not appear to be involved in antimutagenesis. EXERIMENTAL PROCEDURES MaterialsEnzymes-Thermus aquaticus DNA polymerase was from PerkinElmer, and other enzymes used in standard cloning procedures were from Life Technologies, Inc., Stratagene, or U. S. Biochemical Corp. Yeast inorganic pyrophosphata...

Recent studies indicate that disruption of the E-cadherin-mediated cell-cell adhesion system is frequently associated with human cancers of epithelial origin. Reduced levels of both E-cadherin and the associated protein, ␣-catenin, have been reported in human tumors. This report describes the characterization of a human ovarian carcinoma-derived cell line (Ov2008) which expresses a novel mutant form of the ␣-catenin protein lacking the extreme N terminus of the wild-type protein. The altered form of ␣-catenin expressed in Ov2008 cells fails to bind efficiently to ␤-catenin and is localized in the cytoplasm. Deletion mapping has localized the ␤-catenin binding site on ␣-catenin between amino acids 46 and 149, which encompasses the same region of the protein that is deleted in the Ov2008 variant. Restoration of inducible expression of the wild-type ␣-catenin protein in these cells caused them to assume the morphology typical of an epithelial sheet and retarded their growth in vitro. Additionally, the induction of ␣-catenin expression in Ov2008 cells injected into nude mice attenuated the ability of these cells to form tumors. These observations support the classification of ␣-catenin as a growth-regulatory and candidate tumor suppressor gene.The cadherins are a family of transmembrane glycoproteins which mediate Ca 2ϩ -dependent, homotypic cell-cell adhesion in most solid tissues (35, 37). E-cadherin, the predominant cadherin expressed in epithelial cells, localizes to specialized membrane regions known as adherens junctions. Homophilic binding between cadherins on neighboring cells occurs through the direct interaction of their extracellular domains (14). The highly conserved cytoplasmic domain of E-cadherin is associated with a group of proteins collectively known as catenins (␣-, ␤-and ␥-catenin) (19, 24) which have been shown to be essential for cadherin function (8,18,25). Either ␤-catenin or ␥-catenin (plakoglobin) interacts directly with E-cadherin in discrete complexes (4), while ␣-catenin, which does not bind to E-cadherin directly, is subsequently recruited to the complex through its interaction with either ␤-or ␥-catenin (1, 6). ␣-Catenin, which has sequence similarity to vinculin, also contacts actin filaments and consequently serves as a connector between intercellular adhesion and the actin-based cytoskeleton (3,20,25). A fourth cadherin-associated protein, p120 cas , (28) has recently been found associated with cadherin complexes; although it is suggested to play a regulatory role in the assembly of adherens junctions, its precise function remains to be elucidated.A number of recent studies suggest that disruption of the cadherin-based adhesion system may play a role in the progression of human tumors. Immunohistochemical staining has revealed reduced levels of both E-cadherin and ␣-catenin proteins in a variety of human tumors, including tumors of the breast, colon, stomach, esophagus, bladder, and liver (7,11,15,16,21,29,31,32), while higher levels of expression are maintained in the normal epithe...

We show that deletion of a gene of Streptococcus pneumoniae, which we call mutX, confers a mutator phenotype to resistance to streptomycin. Analysis of the DNA sequence changes that occurred in several streptomycin-resistant mutants showed that mutations are unidirectional AT to CG transversions. The mutX gene is located immediately downstream of the previously identified ung gene and genetic evidence suggests that the two genes are co-ordinately regulated. Nucleotide sequence determination reveals that the mutX gene encodes a 17,870 Da protein (154 residues) which exhibits significant homology with the MutT protein of Escherichia coli, a nucleoside triphosphatase (dGTP pyrophosphohydrolase). The mutX gene complements the E. coli mutT mutator phenotype when introduced on a plasmid. Site-directed mutagenesis and analysis of nitrosoguanidine-induced mutT mutants suggest that a small region of high homology between the two proteins (61% identity over 23 residues) is part of the catalytic site of the nucleoside triphosphatase. Computer searching for sequence homology to MutX uncovered a second E. coli protein, the product of orf17, a gene of unknown function located near the ruvC gene. The region of high homology between MutX and MutT is also conserved in this protein, which raises the interesting possibility that the orf17 gene plays some role in determining mutation rates in E. coli. Finally, a small set of proteins, including a family of virus-encoded proteins and two evolutionarily conserved proteins encoded by an antisense transcript from the Xenopus laevis and human bFGF genes, were also found to harbour significant homology to this highly conserved region.