Abstract
Site-directed mutagenesis was carried out at 10 highly conserved polar residues within the C-terminal half of yeast DNA topoisomerase II, which corresponds to the A subunit of bacterial DNA gyrase, to identify amino acid side chains that augment the active site tyrosine Tyr-782 in the breakage and rejoining of DNA strands. Complementation tests show that alanine substitution at Arg-690, Asp-697, Lys-700, Arg-704, or Arg-781, but not at His-735, His-736, Glu-738, Gln-750, or Asn-828, inactivates the enzyme in vivo. Measurements of DNA relaxation and cleavage by purified mutant enzymes show that these activities are abolished in the R690A mutant and are much reduced in the mutants D697A, K700A, R704A, and R781A. When a Y782F polypeptide with a phenylalanine substituting for the active site tyrosine was expressed in cells that also express the R690A polypeptide, the resulting heterodimeric yeast DNA topoisomerase II was found to nick plasmid DNA. Thus in a dimeric wild-type enzyme, Tyr-782 in one protomer and Arg-690 in the other cooperate in trans in the catalysis of DNA cleavage. For the residues D697A, K700A, R704A, and R781A, their locations in the crystal structures of type II DNA topoisomerase fragments suggest that Arg-781 and Lys-700 might be involved in anchoring the 5' and 3' sides of the broken DNA, respectively, and the roles of Asp-697 and Arg-704 are probably less direct.
Highlights
DNA topoisomerases catalyze the breakage of DNA strands via transesterification between an enzyme tyrosyl group and a DNA backbone phosphoryl group, forming a phosphotyrosine link between the two and leaving a deoxyribosyl hydroxyl group on the other end of the broken DNA strand [5,6,7,8,9,10,11]
Selection of Amino Acid Residues for Site-directed Mutagenesis—There are a large number of highly conserved amino acid residues in the type II DNA topoisomerases from a diverse collection of organisms ranging from T-even phage to human
In order to focus on residues that are likely to participate in the catalysis of DNA breakage and rejoining, we applied the same criteria used in a recent study of E. coli DNA topoisomerase I [35]
Summary
GyrA and GyrB, the A and B subunit of bacterial DNA gyrase, respectively; ADPPNP, 5Ј-adenylyl-,␥-imidodiphosphate; E, enzyme; WT, wild type; kb, kilobase pair; HMK, heart muscle kinase. A pair of short DNA helices, each with a four-nucleotide single-stranded extension at a 5Ј end, has been modeled into a DNA-binding site in each half of the (BЈAЈ) protein [24] This structural model is supported by recent protein footprinting experiments in which the effects of DNA binding on the citraconylation of individual lysyl side chains in the yeast enzyme were measured [25]. The crystal structure of a 59-kDa fragment of E. coli GyrA protein, corresponding to the AЈ portion of the 92-kDa yeast fragment, was reported [26] This structure is believed to resemble closely the conformation of the polypeptide in a DNAbound enzyme before the DNA is cleaved by the enzyme [26]. We report mutagenesis analysis of the AЈ subfragment of yeast DNA topoisomerase II
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