THE ZN(II) BINDING MOTIFS OF ESCHERICHIA-COLI DNA TOPOISOMERASE IS PART OF A HIGH-AFFINITY DNA-BINDING DOMAIN

Escherichia coli DNA topoisomerase I binds three Zn(II) with three tet racysteine motifs. Three subclones containing these tetracysteine moti fs were expressed and purified. Subclone ZD1 contained the minimal tet racysteine motifs sequence. A larger subclone ZD2 corresponded to a re gion bordered by two protease sensitive sites. Subclone ZD3 also inclu ded the 14-kDa C-terminal domain that has been shown to bind DNA. Subc lones ZD1 and ZD2 were found to bind one and two Zn(II), respectively, and neither had detectable DNA binding activity. ZD3 could bind three Zn(II) and had higher DNA binding affinity than the 14-kDa C-terminal domain. The complex formed between ZD3 and a single-stranded 31mer co uld be detected by the gel shift assay while the complex formed by the 14-kDa C-terminal domain was not stable under gel electrophoresis con ditions. The three Zn(II) binding motifs appeared to be part of a high -affinity DNA binding domain. (C) 1998 Academic Press.