ISSN:
0887-3585
Keywords:
DNA binding domain
;
etheno-M13 DNA
;
single-stranded DNA affinity chromatography
;
proteolytic fragments
;
truncated topoisomerase
;
protein-DNA interaction
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Medicine
Notes:
Limited digestion of E. coli DNA topoisomerase I with trypsin or papain generated a DNA-binding domain of MW 14,000 corresponding to the carboxyl terminal of the enzyme. This fragment binds to single-stranded DNA agarose as tightly as the intact enzyme. It required around 400 mM NaCl for elution. A truncated topoisomerase that lacks this C-terminal domain was purified. It was eluted from the single-stranded DNA agarose column at around 150 mM NaCl. Although the truncated enzyme could relax negatively supercoiled DNA as efficiently as the intact enzyme at low ionic strength, its processivity was more sensitive to increasing salt concentration. Measurement of binding to fluorescent etheno-M13 DNA also demonstrated that the presence of the C-terminal domain confers higher affinity to DNA for the enzyme.
Additional Material:
5 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/prot.340060307
