Blood coagulation is initiated when tissue factor binds to coagulation factor VIIa to give an enzymatically active complex which then activates factors IX and X, leading to thrombin generation and clot formation. We have determined the crystal structure at 2.0-A degrees resolution of active-site-inhibited factor VIIa complexed with the cleaved extracellular domain of tissue factor. In the complex, factor VIIa adopts an extended conformation. This structure provides a basis for understanding many molecular aspects of the initiation of coagulation.

The 2.8 A resolution crystal structure of the bacteriophage RB69 gp43, a member of the eukaryotic pol alpha family of replicative DNA polymerases, shares some similarities with other polymerases but shows many differences. Although its palm domain has the same topology as other polymerases, except rat DNA polymerase beta, one of the three carboxylates required for nucleotidyl transfer is located on a different beta strand. The structures of the fingers and thumb domains are unrelated to all other known polymerase structures. The editing 3'-5' exonuclease domain of gp43 is homologous to that of E. coli DNA polymerase I but lies on the opposite side of the polymerase active site. An extended structure-based alignment of eukaryotic DNA polymerase sequences provides structural insights that should be applicable to most eukaryotic DNA polymerases.

Nucleic acid polymerases catalyze the formation of DNA or RNA from nucleoside-triphosphate precursors. Amino acid residues in the active site of polymerases are thought to contribute only indirectly to catalysis by serving as ligands for the two divalent cations required for activity or substrate binding. Two proton transfer reactions are necessary for polymerase-catalyzed nucleotidyl transfer: deprotonation of the 3′-hydroxyl nucleophile and protonation of the pyrophosphate leaving group. Using model enzymes representing all four classes of nucleic acid polymerases, we show that the proton donor to pyrophosphate is an active site amino acid residue. The use of general acid catalysis by polymerases extends the mechanism of nucleotidyl transfer beyond that of the well-established two-metal-ion mechanism. The existence of an active-site residue that regulates polymerase catalysis may permit manipulation of viral polymerase replication speed and/or fidelity for virus attenuation and vaccine development.

Several (6), which involved scoring the formation of lung metastases after i.v. injection of melanoma cells into immunodeficient mice (7). These studies showed that human melanoma cell lines expressing high levels of TF, which can initiate coagulation in murine as well as in human plasma, were strongly metastatic and that the metastatic potential of the cell lines could be inhibited by treatment with an anti-TF monoclonal antibody that blocks its procoagulant activity. The conclusion drawn from those results was that one or more products of the coagulation cascade mediate the metastatic effect of TF. In this report we have used a different approach to study the role of TF in promoting metastasis. Four matched sets of cloned human melanoma cell lines expressing either normal or mutant TF molecules were generated by retroviral-mediated transfections, and the metastatic potential of the transfected cells was tested in the SCID mouse model of melanoma metastasis (6 8205The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The single-stranded DNA (ssDNA) binding protein gp32 from bacteriophage T4 is essential for T4 DNA replication, recombination and repair. In vivo gp32 binds ssDNA as the replication fork advances and stimulates replisome processivity and accuracy by a factor of several hundred. Gp32 binding affects nearly every major aspect of DNA metabolism. Among its important functions are: (1) configuring ssDNA templates for efficient use by the replisome including DNA polymerase; (2) melting out adventitious secondary structures; (3) protecting exposed ssDNA from nucleases; and (4) facilitating homologous recombination by binding ssDNA during strand displacement. We have determined the crystal structure of the gp32 DNA binding domain complexed to ssDNA at 2.2 A resolution. The ssDNA binding cleft comprises regions from three structural subdomains and includes a positively charged surface that runs parallel to a series of hydrophobic pockets formed by clusters of aromatic side chains. Although only weak electron density is seen for the ssDNA, it indicates that the phosphate backbone contacts an electropositive cleft of the protein, placing the bases in contact with the hydrophobic pockets. The DNA mobility implied by the weak electron density may reflect the role of gp32 as a sequence-independent ssDNA chaperone allowing the largely unstructured ssDNA to slide freely through the cleft.

The rate-limiting step for nucleotide incorporation in the presteady state for most nucleic acid polymerases is thought to be a conformational change. As a result, very little information is available on the role of active-site residues in the chemistry of nucleotidyl transfer. For the poliovirus RNA-dependent RNA polymerase (3D pol ), chemistry is partially (Mg 2؉ ) or completely (Mn 2؉ ) rate limiting. Here we show that nucleotidyl transfer depends on two ionizable groups with pK a values of 7.0 or 8.2 and 10.5, depending upon the divalent cation used in the reaction. A solvent deuterium isotope effect of three to seven was observed on the rate constant for nucleotide incorporation in the pre-steady state; none was observed in the steady state. Proton-inventory experiments were consistent with two protons being transferred during the rate-limiting transition state of the reaction, suggesting that both deprotonation of the 3 -hydroxyl nucleophile and protonation of the pyrophosphate leaving group occur in the transition state for phosphodiester bond formation. Importantly, two proton transfers occur in the transition state for nucleotidyl-transfer reactions catalyzed by RB69 DNA-dependent DNA polymerase, T7 DNA-dependent RNA polymerase and HIV reverse transcriptase. Interpretation of these data in the context of known polymerase structures suggests the existence of a general base for deprotonation of the 3 -OH nucleophile, although use of a water molecule cannot be ruled out conclusively, and a general acid for protonation of the pyrophosphate leaving group in all nucleic acid polymerases. These data imply an associative-like transition-state structure.general-acid-base catalysis ͉ phosphoryl transfer ͉ two-metal-ion mechanism N ucleic acid polymerases are essential for the maintenance and expression of the genomes of all organisms. All classes of polymerases use the same five-step kinetic scheme for nucleotide incorporation (1-6). The kinetic mechanism for the RNAdependent RNA polymerase (RdRp 1 ) from poliovirus (3D pol ) is shown in Scheme 1. One of the advantages of this system is that once 3D pol assembles onto the primer-template substrate, this complex has a half-life of Ͼ2 h (7), greatly simplifying kinetic analysis. In step one, the enzyme-nucleic acid complex (ER n ) binds the nucleoside triphosphate forming a ternary complex (ER n NTP).Step two involves a conformational change (*ER n NTP) that orients the triphosphate for catalysis. In step three, nucleotidyl transfer occurs (*ER nϩ1 PP i ), followed by a second conformational-change step (ER nϩ1 PP i ) and pyrophosphate release (ER nϩ1 ).Although the sequence of events occurring during the nucleotide-addition cycle is identical for all polymerases, the ratelimiting step appears to be different. In most cases, the first conformational-change step (step two) is rate-limiting (2, 8, 9). In one, chemistry (step three) is rate-limiting (10), and in some (e.g., T4 and RB69 DNA polymerases), the rate-limiting step has not been established. For 3D pol , bot...

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Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. X Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin Kdependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC).Blood coagulation can be initiated by a complex of tissue factor (TF), a membrane-bound glycoprotein, and factor VII, a plasma coagulation factor (for reviews, see refs. 1 and 2). The physiological significance of this extrinsic pathway can be judged by the severe bleeding frequently observed in individuals who are markedly deficient in factor VII (3, 4). In contrast, individuals who have deficiencies or abnormalities in proteins that are involved in the early steps of the intrinsic pathway of coagulation-i.e., high molecular weight kininogen, prekallikrein, and factor XII-are asymptomatic (5). The TF-factor VII complex activates factor IX, a component of the intrinsic pathway, as well as factor X (6). Thus, it is reasonable that the association of TF and factor VII may be the crucial event triggering the initiation of clotting in vivo.The cDNAs for all of the proteins involved in TF-initiated coagulation, with the exception of TF itself, have already been cloned and sequenced (7-13). The TF apoprotein has been purified from both bovine and human sources (14-16). Approximately 50-70% of the amino acid sequence of both species has now been determined, and this has permitted us to select suitable amino acid sequences to serve as a basis for constructing oligonucleotide probes. This in turn has enabled the isolation and characterization of two human placental TF cDNA clones that contain the entire coding region of the mature protein. The nucleotide sequence of these clones, together with amino acid sequence data, has allowed us to formulate a primary structure for the human TF apoprotein §. MATERIALS AND METHODSTF Purification and Sequencing. A monoclonal antibody (17) prepared against human TF that had been purified by using factor VII affinity columns (15) was used for immunoaffinity isolation of TF. Briefly, TF was extracted from human brain or placental tissue acet...