Figure 2.

The presence of Mn²⁺ activates RepB and protects it against thermal inactivation. (A) Activity assays on supercoiled DNA. Samples of RepB unheated, heated to 45°C in the absence of Mn²⁺, or heated to 45 or 70°C in the presence of 20 mM Mn²⁺ were mixed, at the indicated molecular ratios, with 6.7 nM of pMV158 supercoiled DNA and then incubated for 30 min at 37°C in the presence of 20 mM MnCl₂. The supercoiled (SC), closed relaxed circles (Rel), and open circular (OC) plasmid forms were separated by electrophoresis on agarose gels containing ethidium bromide. Images from different parts of the same gel have been grouped and indicated by dividing lines. (B) RepB₆ recognition of the bind DNA measured by EMSA. Unheated or 45°C-heated samples of RepB₆ were mixed, at the indicated molecular ratios, with 0.4 μM of a 42-bp DNA fragment containing the three 11 bp-direct repeats that constitute the bind region of the pMV158 dso. Positions of the free (42-bind) and complexed (C1) DNA are indicated. Images from different parts of the same gel have been grouped and indicated by dividing lines. (C) Supercoiled pMV158 DNA (6.7 nM) was incubated, either at 37°C or at 60°C, with the indicated concentrations of Mn²⁺, in the absence (−) or in the presence (+) of purified RepB₆ (RepB:pMV158 DNA molecular ratio of 20:1).