DNA-binding and DNA helicase activities of the PcrA proteins from <i>M. pneumoniae</i> and <i>M. genitalium</i>.

<p>(A) Binding of the PcrA proteins to 6-FAM-labeled oligonucleotide 1 was tested by EMSA. The PcrA proteins, as well as control protein MBP, were tested at 22 nM, 67 nM and 200 nM, respectively, as indicated from left to right by the triangles above the lanes. Protein was omitted from the reaction shown in lane 1 (‘−’). The positions of the free substrate and the position of protein-DNA complexes (I and II) are indicated at the right-hand side of the gel. Complex I and II are also pointed out by dots next to lanes 13 and 16. ‘ori’ indicates the origin of the gels. (B) Binding of the PcrA proteins and MBP to ds, blunt-ended substrate ‘c’. The experiment was performed similarly as described in (A). (C) Binding of the PcrA proteins and MBP to partially ds substrate ‘a’ (which contains a 3′ 24-nt ss extension). The experiment was executed similarly as described in (A). (D) Binding of the PcrA proteins and MBP to partially ds substrate ‘b’ (which contains a 5′ 24-nt ss extension). The experiment was done in a similar fashion as described in (A). (E) DNA helicase activity of the PcrA proteins on substrate ‘a’. The DNA helicase reactions were performed as described in Materials and Methods. Reactions were carried out in volumes of 10 µl and contained DNA substrate ‘a’ (at 4 nM) and either 0 nM (marked ‘−’, lane 1) or 50 nM of protein (as indicated above the lanes). After the reaction, the samples were deproteinized, electrophoresed through a native 12% polyacrylamide gel, and analyzed by fluorometry. The positions of the substrate and reaction products are indicated at the right-hand side of the gel. (F) DNA helicase activity of the PcrA proteins on substrate ‘b’. The experiment was done in a similar way as in (E). (G) DNA helicase activity of the PcrA proteins on substrate ‘c’. The experiment was performed as described in (E).