Abstract
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 in E. coli AS17 at 42 degreesC, possibly accounting for the conservation of these residues in evolution.
Highlights
DNA topoisomerases catalyze the interconversion of different DNA topological isomers by first forming a covalent enzyme-DNA intermediate via nucleophilic attack of a tyrosine hydroxyl on the DNA phosphodiester linkage
Type IA and type II DNA topoisomerases are linked to the 5Ј-phosphoryl end of the cleaved DNA while type IB DNA topoisomerases are linked to the 3Ј-phosphoryl end
Tyr319 is present in the interface between domains I and III. It has been pointed out [8] that the spatial arrangement of the three acidic residues Asp-111, Asp-113, and Glu-115 in the active site region is similar to the acidic residues that coordinate two divalent cations in the exonuclease catalytic site of Klenow fragment [9]
Summary
Materials—All chemical reagents used were ultrapure or Baker analyzed reagent grade. Solutions were prepared with water first deionized with the Barnstead Nanopure system and passed over a Bio-Rad Chelex 100 resin (100 –200 mesh sodium form) to remove any remaining contaminating metal ions. The labeled DNA was denatured to single strand and incubated with the wild-type and mutant enzymes, and sodium hydroxide was added. After electrophoresis in a 6% DNA sequencing gel, the 5Ј-end-labeled DNA cleavage products were visualized by autoradiography. After incubation of 0.15 g of the labeled oligonucleotide with 0.4 g of the enzyme in 10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 20 mM potassium phosphate at 37 °C for 5 min, the reaction was stopped with the addition of 1% SDS. Gel Mobility Shift Assay—The single-stranded 36-mer 5Ј-TAACCCTGAAAGATTATGCAATGCGCTTTGGGCAAA-3Ј sequence [24] that has the same strong cleavage site as the 9-mer used in the covalent complex formation was labeled at the 5Ј end with T4 polynucleotide kinase and [␥-32P]ATP. The enzyme and dialysis buffer samples were submitted to Quantitative Technologies, Inc., NJ, for Mg(II) content analysis using the inductively coupled plasma method
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