Expression and DNA-binding properties of the 14K carboxyl terminal fragment of Escherichia coli DNA topoisomerase I.

DNA sequence coding for the last 121 amino acids of Escherichia coli topoisomerase I was synthesized by PCR and cloned into a plasmid under the control of the T7 promoter. Induction of T7 RNA polymerase in E. coli carrying the plasmid clone resulted in over-expression of this C-terminal domain fragment previously shown to confer higher DNA binding affinity to the enzyme. Purification to homogeneity was achieved by phosphocellulose and single-stranded DNA agaraose chromatography. Direct interaction between this 14K domain and poly(dA) was demonstrated by UV spectroscopy. Noncovalent complexes formed between this fragment and oligo(dT) 8 and oligo(dT) 16 can also be trapped by photo-crosslinking.