Question

Usingthe HollidayjunctionDNA substratepictured below(50-32Pendlabeled),proposeanassaythatresearchersmay have used to determine RuvA protein binding to the DNA substrate. Propose a modification to the DNA substrate to use as a negative control that demonstrates an aspect of the specificity ofRuvAbinding.(A,B,C,andDarelabelsfortheuniquessDNA fragments that were used to assemble this substrate.)

Answer 1

Researchers might use gel electrophoresis or a fluorescent binding assay to determine the RuvA protein's affinity for a Holliday junction DNA substrate. A negative control with an altered binding site demonstrates the specificity of the RuvA binding.

Step-by-step explanation:

To determine RuvA protein binding to the Holliday junction DNA substrate, researchers may have conducted a binding assay where the DNA substrate is incubated with the RuvA protein and then analyzed using gel electrophoresis to observe the DNA-protein complex. A radioactive phosphorus (32P) end label on the DNA allows for detection of bound complexes. As a non-radioactive alternative, a fluorescent binding assay can be used where the DNA is labeled with a fluorescent tag and the RuvA-DNA complex is detected by fluorescence.

For a negative control, the specific sequence RuvA recognizes can be altered or mutated. This would render the DNA unable to bind RuvA, demonstrating specificity. For instance, this could be achieved by modifying one of the arms of the Holliday junction so that it no longer supports RuvA binding, which would be observable as a lack of fluorescence or radioactive signal when compared to the unmodified substrate.