The role of the Zn(II) binding domain in the mechanism of E. coli DNA topoisomerase I.
BACKGROUND
Escherichia coli DNA topoisomerase I binds three Zn(II) with three tetracysteine motifs which, together with the 14 kDa C-terminal region, form a 30 kDa DNA binding domain (ZD domain). The 67 kDa N-terminal domain (Top67) has the active site tyrosine for DNA cleavage but cannot relax negatively supercoiled DNA. We analyzed the role of the ZD domain in the enzyme mechanism.


RESULTS
Addition of purified ZD domain to Top67 partially restored the relaxation activity, demonstrating that covalent linkage between the two domains is not necessary for removal of negative supercoils from DNA. The two domains had similar affinities to ssDNA. However, only Top67 could bind dsDNA with high affinity. DNA cleavage assays showed that the Top67 had the same sequence and structure selectivity for DNA cleavage as the intact enzyme. DNA rejoining also did not require the presence of the ZD domain.


CONCLUSIONS
We propose that during relaxation of negatively supercoiled DNA, Top67 by itself can position the active site tyrosine near the junction of double-stranded and single-stranded DNA for cleavage. However, the interaction of the ZD domain with the passing single-strand of DNA, coupled with enzyme conformational change, is needed for removal of negative supercoils.
Ahumada
Adriana
A
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY, USA. a_ahumada@msn.com
Tse-Dinh
Yuk-Ching
YC
D001665:Binding Sites; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000378:metabolism*; D004270:DNA, Circular / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008957:Models, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism*
D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D012965:Sodium Chloride; D004247:DNA; D004264:DNA Topoisomerases, Type I; D008274:Magnesium; D015032:Zinc