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The strictly conserved R321 residue in the active site of Escherichia coli topoisomerase I plays a critical role in DNA religation
Author(s) -
Narula Gagandeep,
TseDinh YukChing
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.688.3
DNA topoisomerases are ubiquitous enzymes that overcome topological hindrances encountered by DNA during cellular processes. The enzyme achieves this by cleaving and religating DNA via an active site tyrosine, with the formation of an intermediate covalent cleavage complex. Type IB and type II topoisomerases have served as targets for a number of widely used anticancer and antibacterial drugs. However, due to increasing resistance to these antibacterial drugs, it is essential to identify new drug targets. One such potential target is type IA topoisomerase. However, in order to identify potential interaction sites for the drugs, a detailed understanding of protein‐DNA interactions at the active site of type IA topoisomerases is needed. Previous studies have shown that a conserved arginine residue proximal to the active site tyrosine of type IA topoisomerases is required for the relaxation of supercoiled DNA. Mutants of recombinant Y. pestis topoisomerase I with hydrophobic substitutions at this position exhibited a dominant lethal phenotype, resulting in drastic loss in viability when overexpressed. In depth biochemical analysis of the corresponding E. coli topoisomerase R321 mutants showed that DNA cleavage can take place in the absence of this arginine function if Mg 2+ is present to enhance the interaction of the enzyme with the scissile phosphate. However, DNA religation is inhibited resulting in accumulation of the cleaved covalent intermediate and loss of relaxation activity. These results demonstrate that DNA religation by type IA topoisomerases has a more stringent requirement than DNA cleavage. In addition to the divalent metal ions, the positively charged side chain of this arginine residue is required for the precise positioning of the phosphotyrosine linkage for nucleophilic attack by the 3′‐OH end to result in DNA religation. This research was supported by NIH grants R01 GM054226 and AI069313.