Dissolution of the CBM intermediate created by Blm and Top1.

<p>A. Creation of the CBM, a slower migrating species whose junctions are closer than the starting substrate (DHJS). B. The CBM cannot be dissolved by Top3α alone, but can be in the presence of Blm and Top3α. The results are quantified in the graph below. DNA molecules were analyzed by agarose gel electrophoresis and shown in (A) and (B). C. Same as (B) but using an acrylamide gel to enhance the resolution. The results are quantified in the graph below, and are the results of three independent trials. Error bars indicate standard deviation.</p

Electron microscopy of the CBM intermediate created by Blm and Top1.

<p>A. Categories of molecules observed. B. Quantification of the number of each category present before (DHJS) and after (CBM) reaction with Blm and Top1. C. Comparison of the outer-junction distances of the observed categories of molecules. “Total” is the total length of the molecule, measured from unreacted molecules. The measurements are of the lengths outside of the junctions (since the odd bar shape limits the accuracy of measurement between the junctions). Error bars indicate standard deviation. As expected, the “bar” and “loop” intermediates show an outer-junction distance between full-length (total) and unreacted. D. Comparison of the inter-junction distances of the CBM intermediates. Outer-junction distances were measured and subtracted from the total length. Error bars indicate standard deviation. Although “bar” molecules are on average closer than the “loop” molecules, they are within error of each other. E. Distribution of measured inter-junction distances shown in (D).</p

Occupancy of enzymes on the dHJ substrate, as visualized by electron microscopy.

<p>Both Top3α and Blm prefer to bind at the junction site of the dHJ substrate.</p

Electron microscopy of proteins on the dHJ substrate.

<p>A. Top3α bound to junction and non-junction sites. B. Blm bound at junctions and forming larger complexes with multiple DNA monomers. Enzymes were incubated with the DNA in dissolution buffer, in the absence of ATP to prevent helicase activity.</p

Testing for migration of the HJ using the mismatch dHJ substrate.

<p>A diagram of the reaction is at the top. Complete dHJ dissolution is indicated by the appearance of two characteristic bands after digestion with just BamHI (first lane of each set). If migration is successful, digestion with BamHI and NheI (second lane in each set) or BamHI and SphI (third lane in each set) will result in three characteristic bands. Migration can occur even if dissolution does not. Although some background is evident, it is clear that substantial migration occurs in the presence of either topoisomerase, but dissolution occurs only with Top3α.</p