William H. Konigsberg

Crystal Structure of a pol α Family Replication DNA Polymerase from Bacteriophage RB69

Abstract The 2.8 A resolution crystal structure of the bacteriophage RB69 gp43, a member of the eukaryotic pol α 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 β, one of the three carboxylates required for nucleotidyl transfer is located on a different β 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.

A micromethod for complete removal of dodecyl sulfate from proteins by ion-pair extraction

Abstract Methods are presented for the complete removal of dodecyl sulfate from proteins. Themethods utilize the extraction of dodecyl sulfate anions as ion pairs with triethylammonium or tributylammonium cations into an organic solvent. The protein is insoluble in the organic solvent and is recovered as a precipitate. The methods are applicable to microgram as well as milligram amounts of protein. In all cases studied, the recovery of protein ranges from 70 to 100%. The recovered protein is suitable for N-terminal Edman degradation, tryptic peptide mapping, and amino acid analysis and can be renatured to regain enzymatic activity and antigenicity.

Nucleic acid polymerases use a general acid for nucleotidyl transfer.

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 that are 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.

Tumor necrosis factor enhances expression of tissue factor mRNA in endothelial cells

We have examined the effect of recombinant tumor necrosis factor on the expression of tissue factor activity and tissue factor mRNA levels in vascular endothelial cells. Following exposure of human umbilical vein endothelial cells to this cytokine, the appearance of tissue factor procoagulant activity was detected following cell disruption, and was maximal at 6 hours. Northern blot analysis of cytokine-treated cells demonstrated a similar increase in the synthesis of tissue factor mRNA, followed by a gradual decline to the basal level by 18 hours. Cycloheximide by itself induced the accumulation of high levels of tissue factor mRNA in these cells. This result suggests that the proteins necessary for transcription of the tissue factor gene are present in the endothelial cell prior to cytokine stimulation, and synthesis of the tissue factor mRNA may be controlled, in part, by a labile repressor protein.

[13] Reduction of disulfide bonds in proteins with dithiothreitol.

Publisher Summary The cleavage of disulfide bridges is of importance in both functional and structural studies of proteins. Of the three available methods for accomplishing this—namely, oxidation, sulfitolysis and reduction, the third procedure is often the method of choice since the first method suffers from lack of reversibility as well as undesirable side reactions, and the second from the difficulty in achieving complete reaction. While β-mercaptoethanol has been extensively used for both selective and complete reduction of disulfide bridges, it can be replaced by dithiothreitol (DTT). This reagent can be used at a much lower concentration than β-mercaptoethanol by virtue of its lower oxidation-reduction potential and its resistance to air oxidation compared to β-mercaptoethanol. This can be of particular importance when radioactive alkylating agents are to be employed, since only a very small molar excess of DTT is required for complete reduction and therefore only minimal quantities of radioactive material need be wasted in alkylating excess reducing agent.

Cloning and expression of human tissue factor

Tissue factor is a membrane protein that plays an essential role in the initiation of blood coagulation. When exposed to the circulation, tissue factor interacts with the serine protease factor VII, and the complex triggers fibrin clot formation by activating both factors IX and X of the coagulation cascade. This report describes the cloning and expression of the complementary DNA (cDNA) for human tissue factor. The cDNA encodes a protein of 263 amino acids preceded by a 32 amino acid signal peptide. The predicted protein sequence contains a potential hydrophobic membrane anchoring domain at its carboxy terminus, and bears no significant homology to any other known protein. Tissue factor mRNA of 2400 nucleotides was detected in adipose, adrenal, small intestine and a number of other tissues by Northern blot hybridization analysis. In order to confirm the identity of the cDNA, an expression vector containing the cloned cDNA was used to transfect cultured mammalian cells. These cells produced active tissue factor which was assayed using purified factors VII and X.

Single-molecule and ensemble fluorescence assays for a functionally important conformational change in T7 DNA polymerase

We report fluorescence assays for a functionally important conformational change in bacteriophage T7 DNA polymerase (T7 pol) that use the environmental sensitivity of a Cy3 dye attached to a DNA substrate. An increase in fluorescence intensity of Cy3 is observed at the single-molecule level, reflecting a conformational change within the T7 pol ternary complex upon binding of a dNTP substrate. This fluorescence change is believed to reflect the closing of the T7 pol fingers domain, which is crucial for polymerase function. The rate of the conformational change induced by a complementary dNTP substrate was determined by both conventional stopped-flow and high-time-resolution continuous-flow fluorescence measurements at the ensemble-averaged level. The rate of this conformational change is much faster than that of DNA synthesis but is significantly reduced for noncomplementary dNTPs, as revealed by single-molecule measurements. The high level of selectivity of incoming dNTPs pertinent to this conformational change is a major contributor to replicative fidelity.

Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases

The rate-limiting step for nucleotide incorporation in the pre-steady 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 (3Dpol), chemistry is partially (Mg2+) or completely (Mn2+) rate limiting. Here we show that nucleotidyl transfer depends on two ionizable groups with pKa 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.

Formation of tissue factor–factor VIIa–factor Xa complex promotes cellular signaling and migration of human breast cancer cells

Summary.  Tissue factor (TF) is a transmembrane glycoprotein that initiates blood coagulation when complexed with factor (F)VIIa. Recently, TF has been shown to promote cellular signaling, tumor growth, angiogenesis, and metastasis. In the present study, we examined the pathway by which TF–FVIIa complex induces cellular signaling in human breast cancer cells using the Adr‐MCF‐7 cell line. This cell line has high endogenous TF expression as measured by flow cytometry and expression of protease‐activated receptors 1 and 2 (PAR1 and PAR2) as determined by reverse transcriptase‐polymerase chain reaction analysis. Both PAR1 and PAR2 are functionally active as determined by induction of p44/42 mitogen‐activated protein kinase (MAPK) phosphorylation using specific agonist peptides. We found that MAPK phosphorylation in this cell line was strongly induced by the combination of FVIIa and factor (F)X, but not by FVIIa alone at a concentration of FVIIa that approaches physiological levels. Induction of MAPK phosphorylation involved the formation of TF–FVIIa–FXa complex and occurred by a pathway that did not require thrombin formation, indicating a critical role for FXa generation. In addition, induction of MAPK phosphorylation was found to be independent of PAR1 activation. We then examined whether TF–FVIIa complex formation could promote tumor cell migration using a modified Boyden chamber chemotaxis assay. The combination of FVIIa and FX, but not FVIIa alone, strongly induced migration of tumor cells by a pathway that probably involves PAR2, but not PAR1 activation. MAPK phosphorylation was found to be required for the induction of cell migration by the combination of FVIIa and FX. These data suggest that TF–FVIIa‐mediated signaling in human breast cancer cells occurs most efficiently by formation of the TF–FVIIa–FXa complex. One of the physiological consequences of this signaling pathway is enhanced cell migration that is probably mediated by PAR2, but not PAR1 activation.

The amino acid sequence of a DNA binding protein, the gene 5 product of fd filamentous bacteriophage

The F-specific filamentous bacterial viruses such as fd, fl and Ml3 synthesize single-stranded circular viral DNA on a duplex DNA intermediate [ 1 ] . Synthesis of this single-stranded DNA requires the products of viral gene 2 [2,3] and gene 5 [4] as well as rifampitin-sensitive funtion [5] . The gene 5 product (5~) a protein having a molecular weight of 9830 daltons, forms a complex with intracellular single-stranded DNA preventing the synthesis of a complementary DNA strand [6] . During maturation of the virus, the 5p protein is completely displaced from the DNA by the coat protein associated with the bacterial membrane [7]. Because of the importance of 5p in DNA replication and phage maturation, we have determined the primary structure of 5p, as a necessary step prior to studying the molecular details of the interaction of 5p with DNA in solution and in crystals. 80 mg of 5p could be obtained from 100 g of cells. The purity of 5p was about 95% as shown by polyacrylamide gel electrophoresis [ 111.