title: Iron inhibits Escherichia coli topoisomerase I activity by targeting the first two zinc-binding sites in the C-terminal domain.
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.
authors.affiliation: Key Laboratory of Laboratory Medicine, Ministry of Education of China, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, 325035, China.
authors.forename: Wu
authors.initials: W
authors.lastname: Wang
authors.forename: Xiaolu
authors.initials: X
authors.lastname: Su
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Wang
authors.forename: Juanjuan
authors.initials: J
authors.lastname: Yang
authors.forename: Ting
authors.initials: T
authors.lastname: Zhang
authors.forename: Maofeng
authors.initials: M
authors.lastname: Wang
authors.forename: Rugen
authors.initials: R
authors.lastname: Wan
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Tan
authors.forename: Jianxin
authors.initials: J
authors.lastname: Lu
mesh_terms: D001665:Binding Sites; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D007501:Iron / Q000378:metabolism / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D059004:Topoisomerase I Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D015032:Zinc / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D059004:Topoisomerase I Inhibitors; D007501:Iron; D004264:DNA Topoisomerases, Type I; D015032:Zinc
keywords: Escherichia coli topoisomerase I; iron binding; topoisomerase activity; zinc-binding motif