You identify a new topoisomerase from a thermophilic bacterium and want to know by what increment it changes the linking number of DNA.

1.

a.      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.

b.     Lets assume you passed on answering (a.) and left it to a co-worker in the lab. 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 provided at the end of this exam.

2.

a.     Draw a diagram of the E. coli replication fork with at least 5 of the proteins/protein complexes that serve key functions in replication of the E. coli chromosome. Provide a brief description of the function of each of these proteins or protein complexes.

b.     Draw a scheme for the regulation of origin firing of the E. coli origin of replication, including important features of the sequences that make up the origin of replication, and key proteins that initiate origin firing and prevent re-initiation soon after initiation of replication.

c.      The structure of DNA as deduced by Watson and Crick immediately suggested that the strands are copied by pulling them apart, and using the single-stranded molecules as templates for synthesis of new strands. This proposal was termed the “semiconservative” model of replication, since one half of each new double helix was from the parental molecule, and one half was newly synthesized. Briefly discuss one alternative model for the mechanism of DNA synthesis, and describe the experimental design and outcome used to deduce which model is the correct one.