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Iron inhibits E scherichia coli topoisomerase I activity by targeting the first two zinc‐binding sites in the C‐terminal domain
Author(s) -
Wang Wu,
Su Xiaolu,
Wang Xiaobing,
Yang Juanjuan,
Zhang Ting,
Wang Maofeng,
Wan Rugen,
Tan Guoqiang,
Lu Jianxin
Publication year - 2014
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2542
Subject(s) - binding site , topoisomerase , chemistry , dna , zinc finger , binding domain , zinc , biochemistry , plasma protein binding , mutagenesis , escherichia coli , biophysics , microbiology and biotechnology , biology , mutation , gene , transcription factor , organic chemistry
Abstract Escherichia coli DNA topoisomerase I (TopA) contains a 67 kDa N‐terminal catalytic domain and a 30 kDa C‐terminal zinc‐binding region (ZD domain) which has three adjacent tetra‐cysteine zinc‐binding motifs. Previous studies have shown that E. coli TopA can bind both iron and zinc, and that iron binding in TopA results in failure to unwind the negatively supercoiled DNA. Here, we report that each E. coli TopA monomer binds one atom of iron via the first two zinc‐binding motifs in ZD domain and both the first and second zinc‐binding motifs are required for iron binding in TopA. The site‐directed mutagenesis studies further reveal that while the mutation of the third zinc‐binding motif has very little effect on TopA's activity, mutation of the first two zinc‐binding motifs in TopA greatly diminishes the topoisomerase activity in vitro and in vivo , indicating that the first two zinc‐binding motifs in TopA are crucial for its function. The DNA‐binding activity assay and intrinsic tryptophan fluorescence measurements show that iron binding in TopA may decrease the single‐stranded (ss) DNA‐binding activity of ZD domain and also change the protein structure of TopA, which subsequently modulate topoisomerase activity.