Figure 7.
(A and B) Analysis of DNA-binding activity of the BLM642–1290 and mutant BLMs using electrophoretic mobility shift assay. One nanomole of 5'-32P-labelled 36-mer ssDNA (oligonucleotide C, Table 1) was incubated at room temperature for 20 min with various concentrations of protein between 2.5 and 160 nM in unwinding buffer. Bound and free DNA were separated by electrophoresis on a non-denaturing 15% polyacrylamide gel and detected by autoradiography. (C and D) The anisotropy-based DNA-binding isotherms of wild-type and mutant BLM proteins. Fluorescence anisotropy values were determined as a function of enzyme concentrations for 21-mer ssDNA substrates (oligonucleotide D, Table 1). Two nanomolar fluorescein-labelled DNA was titrated with various amounts of RecQ under conditions as described in Materials and Methods section. The solid lines represent the best fits of the data to Equation (2). The determined values of dissociation constant per binding site (Kd/N) are summarized in Table 2.