Chang-Xi Zhu

Effect of Mg(II) Binding on the Structure and Activity ofEscherichia coli DNA Topoisomerase I

Escherichia coli DNA topoisomerase I requires Mg(II) as a cofactor for the relaxation of negatively supercoiled DNA. Mg(II) binding to the enzyme was shown by fluorescence spectroscopy to affect the tertiary structure of the enzyme. Addition of 2 mm MgCl2 resulted in a 30% decrease in the maximum emission of tryptophan fluorescence of the enzyme. These Mg(II)-induced changes in fluorescence properties were reversible by the addition of EDTA and not obtained with other divalent cations. After incubation with Mg(II) and dialysis, inductively coupled plasma (ICP) analysis showed that each enzyme molecule could form a complex with 1–2 Mg(II) bound to each enzyme molecule. Such Mg(II)·enzyme complexes were found to be active in the relaxation of negatively supercoiled DNA in the absence of additional Mg(II). Results from ICP analysis after equilibrium dialysis and relaxation assays with limiting Mg(II) concentrations indicated that both Mg(II) binding sites had to be occupied for the enzyme to catalyze relaxation of negatively supercoiled DNA.

Site-directed Mutagenesis of Conserved Aspartates, Glutamates and Arginines in the Active Site Region of Escherichia coli DNA Topoisomerase I

To catalyze relaxation of supercoiled DNA, DNA topoisomerases form a covalent enzyme-DNA intermediate via nucleophilic attack of a tyrosine hydroxyl group on the DNA phosphodiester backbone bond during the step of DNA cleavage. Strand passage then takes place to change the linking number. This is followed by DNA religation during which the displaced DNA hydroxyl group attacks the phosphotyrosine linkage to reform the DNA phosphodiester bond. Mg(II) is required for the relaxation activity of type IA and type II DNA topoisomerases. A number of conserved amino acids with acidic and basic side chains are present near Tyr-319 in the active site of the crystal structure of the 67-kDa N-terminal fragment of Escherichia coli DNA topoisomerase I. Their roles in enzyme catalysis were investigated by site-directed mutation to alanine. Mutation of Arg-136 abolished all the enzyme relaxation activity even though DNA cleavage activity was retained. The Glu-9, Asp-111, Asp-113, Glu-115, and Arg-321 mutants had partial loss of relaxation activity in vitro. All the mutants failed to complement chromosomal topA mutation inE. coli AS17 at 42 °C, possibly accounting for the conservation of these residues in evolution.

Systematic investigation of crystallization parameters for protein-nucleic acid complexes: application to an active truncated form ofEscherichia coli topoisomerase I

Abstract An experimental strategy to systematically screen possible crystallization parameters for protein:oligonucleotide complexes is described and successfully used in the cocrystallization of a truncated form of Escherichia coli topoisomerase I with an oligo(thymidylic acid) polymer of 8 bases. Crystals are orthorhombic ( a = 65.9A˚, b =74.0A˚, and c = 140.2A˚) and diffract to at least 3A˚resolution.