Role of recombinational repair in sensitivity to an antitumour agent that inhibits bacteriophage T4 type II DNA topoisomerase.

The bacteriophage T4-encoded type II DNA topoisomerase is the major target for the antitumour agent m-AMSA (4'-(9-acridinylamino)methanesulphonm-ansidide) in phage-infected bacterial cells. Inhibition of the purified enzyme by m-AMSA results in formation of a cleavage complex that contains the enzyme covalently attached to DNA on both sides of a double-strand break. In this article, we provide evidence that this cleavage complex is responsible for inhibition of phage growth and that recombinational repair can reduce sensitivity to the antitumour agent, presumably by eliminating the complex (or some derivative thereof). First, topoisomerase-deficient mutants were shown to be resistant to m-AMSA, indicating that m-AMSA inhibits growth by inducing the cleavage complex rather than by inhibiting enzyme activity. Second, mutations in several phage genes that encode recombination proteins (uvsX, uvsY, 46 and 59) increased the sensitivity of phage T4 to m-AMSA, strongly suggesting that recombination participates in the repair of topoisomerase-mediated damage. Third, m-AMSA stimulated recombination in phage-infected bacterial cells, as would be expected from the recombinational repair of DNA damage. Finally, m-AMSA induced the production of cleavage complexes involving the T4 topoisomerase within phage-infected cells.

Disruption of a topoisomerase-DNA cleavage complex by a DNA helicase.

The type II DNA topoisomerases are targets for a variety of chemotherapeutic agents, including the antibacterial quinolones and several families of antitumor drugs. These agents stabilize an enzyme-DNA cleavage complex that consists of the topoisomerase covalently linked to the 5' phosphates of a double-stranded DNA break. Although the drug-stabilized cleavage complex is readily reversible, it can result in cell death by a mechanism that remains uncertain. Here we demonstrate that the action of a DNA helicase can convert the cleavage complex into a nonreversible DNA break by displacing DNA strands from the complex. Formation of a nonreversible DNA break, induced by a DNA helicase, could explain the cytotoxicity of these topoisomerase poisons.

Thermodynamics of active-site ligand binding to Escherichia coli glutamine synthetase.

Active-site ligand interactions with dodecameric glutamine synthetase from Escherichia coli have been studied by calorimetry and fluorometry using the nonhydrolyzable ATP analogue 5'-adenylyl imidodiphosphate (AMP-PNP), L-glutamate, L-Met-(S)-sulfoximine, and the transition-state analogue L-Met-(S)-sulfoximine phosphate. Measurements were made with the unadenylylated enzyme at pH 7.1 in the presence of 100 mM KCl and 1.0 mM MnCl2, under which conditions the two catalytically essential metal ion sites per subunit are occupied and the stoichiometry of active-site ligand binding is equal to 1.0 equiv/subunit. Thermodynamic linkage functions indicate that there is strong synergism between the binding of AMP-PNP and L-Met-(S)-sulfoximine (delta delta G' = -6.4 kJ/mol). In contrast, there is a small antagonistic effect between the binding of AMP-PNP and L-glutamate (delta delta G' = +1.4 kJ/mol). Proton effects were negligible (less than or equal to 0.2 equiv of H+ release or uptake/mol) for the different binding reactions. The binding of AMP-PNP (or ATP) to the enzyme is entropically controlled at 303 K with delta H = +5.4 kJ/mol and delta S = +150 J/(K.mol). At 303 K, the binding of L-glutamate (delta H = -22.2 kJ/mol) or L-Met-(S)-sulfoximine [delta H = -45.6 kJ/mol with delta Cp approximately equal to -670 +/- 420 J/(K.mol)] to the AMP-PNP.Mn.enzyme complex is enthalpically controlled with opposing delta S values of -29 or -46 J/(K.mol), respectively. The overall enthalpy change is negative and the overall entropy change is positive for the simultaneous binding of AMP-PNP and L-glutamate or of AMP-PNP and L-Met-(S)-sulfoximine to the enzyme. For the binding of the transition-state analogue L-Met-(S)-sulfoximine phosphate (which inactivates the enzyme by blocking active sites), both enthalpic and entropic contributions also are favorable at 303 K [delta G' approximately equal to -109 and delta H = -54.8 kJ/mol of subunit and delta S approximately equal to +180 J/(K.mol)].

Eukaryotic initiation factor 4D. Purification from human red blood cells and the sequence of amino acids around its single hypusine residue.

Eukaryotic initiation factor 4D (eIF-4D) was purified from human red blood cells by a simple 5-step procedure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the preparations of eIF-4D were composed of variable amounts of two closely migrating forms of the factor, each of which contained a single residue of the unique amino acid hypusine. The structural similarity of the two forms of human eIF-4D was evidenced by the indistinguishable patterns of radioactivity on peptide maps of tryptic digests prepared from radioiodinated samples. A peptide containing the single hypusine residue was readily isolated from a tryptic digest of human eIF-4D by virtue of its high positive charge and hydrophilic character. Amino acid sequence determination on this peptide revealed the following primary structure around hypusine: Thr-Gly-hypusine-His-Gly-His-Ala-Lys.

The use of 4-(2-pyridylazo)resorcinol in studies of zinc release from Escherichia coli aspartate transcarbamoylase.

The metallochromic indicator 4-(2-pyridylazo)resorcinol (PAR) has been used at pH 7.0 to monitor the mercurial-promoted Zn2+ release from Escherichia coli aspartate transcarbamoylase and Zn2+ uptake by regulatory dimers upon displacement of the mercurial reagent with 2-mercaptoethanol. The release of Zn2+ (as reflected by a yellow to orange color change in PAR solutions) is linked to dissociation of the enzyme since the six Zn2+ bonding domains stabilize catalytic and regulatory chain contacts; the rebinding of Zn2+ produces enzyme assembly and a corresponding decrease in the amount of PAR-Zn2+ complex. Using greater than 10-fold PAR to free Zn2+ at pH 7.0, delta epsilon = 6.6 +/- 0.2 X 10(4) M-1 cm-1 at 500 nm (20 degrees C) for (PAR)2Zn2+ complex formation (beta'2 approximately equal to 10(12) M-1). In kinetic studies at pH 7.0, PAR (10(-4) M) has been used to measure the instantaneous concentration of Zn2+ released from micromolar quantities of protein; second-order k = 2 X 10(7) M-1 s-1 for forming the 1:1 PAR:Zn2+ complex. These properties of PAR-Zn2+ interactions make PAR a generally useful reagent for studying Zn2+ release from proteins.

Mercurial-promoted Zn2+ release from Escherichia coli aspartate transcarbamoylase.

The release of Zn2+ from aspartate transcarbamoylase (ATCase; c6r6) upon challenge by p-hydroxymercuriphenylsulfonate (PMPS) has been studied using the sensitive, high-affinity metallochromic indicator 4-(2-pyridylazo)resorcinol at pH 7.0. When the--SH group of each catalytic (c) chain is protected, 1 Zn2+ is released for every 4 eq of PMPS added to ATCase during titration of the 24--SH groups of regulatory (r) chains. Moreover, the release of Zn2+ is a linear function of PMPS added, indicating that the rate-limiting step in Zn2+ release is mercurial attack on the 1st of the 4 r--SH groups bonded tetrahedrally to Zn2+ in an r chain near c:r contacts. Dissociation of ATCase is linked to Zn2+ release and mercaptide formation; e.g. upon addition of 4 eq of PMPS to ATCase in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer, 1/6th of ATCase is dissociated to c3 and r2 subunits at approximately 83% of the rate of Zn2+ release, with no accumulation of the c6r4 intermediate as is observed in KPO4 buffer. Adding less than or equal to 4 PMPS/ATCase, the release of Zn2+ is first-order in [PMPS] and is virtually independent of [ATCase] with an activation energy of 18 kcal/mol. With large excesses of PMPS, stopped-flow traces show a lag period followed by pseudo first-order release of Zn2+ from ATCase and the reaction order in [PMPS] = approximately 1.3. Under these conditions, PMPS has a chaotropic effect on ATCase; the activation energy for Zn2+ release is much lower than that obtained with limiting PMPS and is increased by the presence of phosphate or active-site ligand from 6.6 to approximately 12 kcal/mol. A reasonable explanation of the observed kinetic data is that the organomercurial reagent binds reversibly to nitrogenous side chain groups in an ATCase molecule prior to the rate-limiting reaction with a sulfhydryl group.