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.
Zhu
C X
CX
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla 10595, USA.
Samuel
M
M
Pound
A
A
Ahumada
A
A
Tse-Dinh
Y C
YC
D017123:Bacteriophage T7; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D004264:DNA Topoisomerases, Type I / Q000096:biosynthesis* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D011061:Poly A / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D017931:DNA Primers; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D011061:Poly A; C015465:poly(dA); D004247:DNA; D004264:DNA Topoisomerases, Type I