The difference in the sensitivities of the two bacterial strains to CQ is relatively modest, indicating that there are other targets for CQ in the bacteria. the specificity of action, is influenced by the DNA-binding characteristics of the inhibitor, and the inhibition is noncompetitive with respect to NAD+. The arylamino compounds appear to target eubacterial DNA ligase in vivo, since a Lig? strain that has been rescued with the ATP-dependent T4 DNA ligase is less sensitive than the parental strain. DNA ligation is an important step in a number of cellular processes, including replication, recombination, and repair of damaged DNA (8, 14, 15). As a consequence, DNA ligase is an essential enzyme in every organism. Its key role in living organisms is evidenced by the facts that eukaryotic cells encode up to five isoenzymes (40) and that many viruses encode their own DNA ligase (13, 28). Studies of conditionally lethal and null mutants of DNA ligase genes (2, 8, 22) indicate that the single DNA ligase of bacteria is an essential enzyme. In eukaryotic systems, disruption of DNA ligase. We also demonstrate that the endogenous eubacterial DNA ligase can be targeted in vivo by some of these compounds, thereby providing the first LY-3177833 practical demonstration that DNA ligase inhibitors do indeed represent a novel class of agents with antimicrobial activity. MATERIALS AND METHODS Cells and chemicals. The LT2 wild-type strain of (22), its DNA ligase-minus (null) derivative which was constructed in the presence of the T4 Lig+ plasmid pBR313/598/8/1b (TT15151 [43]), the conditional lethal (8) mutant strain of (TT6508), and its derivative (UAG, Ty8) pBR313/598/8/1b (TT8352), harboring the gene for T4 DNA ligase (T4 Lig+ [43]), were all kindly provided by J. R. Roth (University of Utah, Salt Lake City). Doxorubicin and hydroxychloroquine were gifts from Farmitalia-Carlo Erba (Milan, Italy) and Sanofi Winthrop (Newcastle upon Tyne, United Kingdom), respectively. The 2-amino-5-diethylaminopentane was purchased from Aldrich, Castle Hill, Australia. [3H]dTTP was obtained from Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, United Kingdom. Mefloquine was kindly donated by Hoffmann-La Roche, Basel, Switzerland. DNA ligase and exonuclease III were from Boehringer Mannheim, Castle Hill, Australia. Human recombinant DNA ligase I was purified as described elsewhere (7). Due to the difficulty of isolating large quantities of active LY-3177833 human DNA ligase, the more extensive studies of inhibition of ATP-dependent ligase were performed with the T4 DNA ligase. Nutrient broth and nutrient agar were LY-3177833 supplied by Oxoid Pty. Ltd., West Heidelberg, Australia. All other drugs and chemicals were from Sigma Chemical Co. DNA ligase assays. DNA joining activity was measured by determining the extent to which nicked [3H] poly(dA-dT) became resistant to degradation by exonuclease III, using a modification of the method of Modrich and Lehman (18). The DNA ligase reaction mixture (50 l) contained 30 mM Tris-HCl (pH 8.0), 4 mM MgCl2, 1.2 mM EDTA, 1 mM dithioerythritol, 0.026 mM NAD, 50 g of bovine serum albumin/ml, and 2 nmol of [3H]poly(dA-dT) (ca. 60 cpm/pmol). The bacteriophage T4 DNA ligase and human recombinant DNA ligase I reaction mixtures (50 l) contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 1 mM dithioerythritol, 1 mM ATP, and 2 nmol of [3H]poly(dA-dT) (ca. 60 cpm/pmol). Upon addition of enzyme, the reaction mixture was incubated for 30 min at 37C, then boiled for 3 min. After the reaction mixture was chilled, 50 l of a solution containing 80 mM Tris-HCl (pH 8.0), 5 mM dithioerythritol, 30 g of bovine serum albumin/ml, and 15 U of exonuclease III was added. After NUDT15 incubation for 30 min at 37C, 80-l samples were spotted onto Whatman GF/C filters and the filters were batch washed three times with 5% (wt/vol).
