Figure 3.

Translocation on χ-substrate DNA at high HsdR concentrations. (A) Structure of the HJ region of the χ-intermediate. The continuous 3′–5′ translocated strand is indicated as a thicker line. Note, the HJ is mobile within the homologous regions bounded by the cer sites (yellow triangles). Translocation by EcoR124I away from the HJ does not interfere with the reaction and is not considered. (B) Representative agarose gel showing separation of the DNA species produced by EcoNI/ScaI digestion and bound by TFO_south. DNA (5 nM) was pre-bound with ³²P-labelled TFO_south as described (11), and then treated with 90 nM HsdR and 40 nM MTase in buffer R for the times shown. Images were captured using a Molecular Dynamics Typhoon 9200 PhosphorImager and quantified using ImageQuant software. Note the rapid displacement of TFO_south from the linear DNA. (C) TFO-binding to the χ-species was calculated relative to the zero time point sample, which was set up separately. Because the χ species represent only a fraction of the total sample DNA, experimental variation in sample composition leads to the percentage of bound TFO on the HJ substrate exceeding 100% in some cases. Each triplex was analysed in a separate reaction. Data represents the average of ≥2 independent experiments. The inset shows the first 5 min of the reactions.