Question

1. You identify a new topoisomerase from a thermophilic bacterium and want to know by what increment it changes the linking number of DNA. Outline an experiment by which you could determine whether the enzyme changes Lk in increments of 1, 2 (or more.) Please consider how you would generate the substrate for the enzyme assay, how you would conduct the assay, and how you would interpret the results.
2. Lets assume you passed on answering (a.) and left it to a co-worker in the la She successfully designs and executes the experiment and determines that the correct answer is “2”. Please provide a schematic diagram outlining the mechanism of action of this enzyme. Is there likely to be a covalent enzyme intermediate? If so, please draw its structure, referring to the amino-acid and nucleotide structures.

Answer 1

Q.A ) The enzymes Topoisomerases control the underwinding or overwinding of DNA. Hence, they introduce or remove supercoils of DNA, for example during DNA replication of transcription the DNA would get strained due to supercoils and these are removed by the Topoisomerases and make it available for that particular process.

The assay to study the new Topoisomerase is as follows:

Take relaxed DNA and sc DNA as substrates and then take five tubes. In one tune take only relaxed DNA without any enzyme as a control. In tube-2 take sc-DNA with enzyme as second control. Now in tube 3 to 5 take relaxed DNA with enzyme and incubate them for different time intervals. After then run the gel and check for number of bands obtained.

Relaxed DNA -3 SC DNA-> -7 Lane 1: relaxed DNA substrate Lane 2: scDNA+topoisomerase l Lane 3: relaxed DNA+gyrase (10sec) Lane 4: relaxed DNA+ gyrase (20sec) Lane 5: relaxed DNA+gyrase (40sec)

So, in lane-1 the relaxed DNA doesnot have any supercoils, hence it has more surface area and it stooped at the top of the gel. In lane-2 to -3 these enzymes introduced supercoils in DNA; hence at each stage the DNA getting down due to increasing negative supercoiling and linking number (LK) with the time. Thus, due to the presence of negative supercoils in DNA the gel would have different types of ladders.