Protein DNA Dance

Dance Your Ph.D. 2010 Video: "DNA Structural Selectivity of Binding by the Pol I DNA Polymerases from Escherichia coli and Thermus aquaticus", Andy J. Wowor, 2009, Louisiana State University.

The study of DNA polymerases is important because DNA replication is one of the fundamental processes occurring in living organisms and DNA polymerases are responsible for the replication of genetic information in any organism. DNA polymerases are also used as a tool in the polymerase chain reaction (PCR) technique in biotechnological applications such as cloning, gene analysis/fingerprinting, and detection of diseases.

For my Ph.D. research, I studied the DNA structure selectivity of polymerases using fluorescence anisotropy. Understanding the substrate selection by DNA polymerase I is important for characterizing the balance between DNA replication and repair for this enzyme in vivo. Klenow and Klentaq, the "large fragments" of the DNA polymerase I from Escherichia coli (37°C) and Thermus aquaticus (75°C), are considered functional homologues because of their sequence and structural similarities. Klentaq, however, does not have a functional "proofreading" site. Klenow exhibits significant differences in binding to pt-DNA versus ds-DNA, while Klentaq does not, suggesting that Klenow and Klentaq discriminate between these two DNA structures differently.

Note: What is the difference between primer-template DNA (pt-DNA) and blunt-end DNA (ds-DNA)? One strand of pt-DNA is longer than the other one while both strands of ds-DNA have the same length.

Fun facts: In 1885, Theodor Escherich discovered a mesophilic bacterium, Escherichia coli, in the human colon. Arthur Kornberg and colleagues isolated DNA polymerase I from Escherichia coli in 1955. T. D. Brock and H. Freeze discovered a thermophilic bacterium, Thermus aquaticus, in 1969 at a hot spring in Yellowstone National Park. The DNA polymerase I from Thermus aquaticus (Taq polymerase) was first isolated by Chien and colleagues in 1976.

Please watch the dance before reading further! I hope that you enjoy it!!!



Spoilers:

1. Two girls on the left side of the screen represent pt-DNA while two girls on the right side of the screen represent ds-DNA. One strand of pt-DNA is longer than the other one while both strands of ds-DNA have the same length. The man on the right plays a scientist who is gathering data using an instrument.

2. The movements of these DNA molecules were inspired by Doris Humphrey's "fall and recovery" principle. Along with Martha Graham (who developed the "contraction and release" principle), Humphrey was a modern dance pioneer who studied, taught and performed dance/choreography at the Denishawn School of Dancing and Related Arts.

3. Part I: Klenow-DNA Interaction. A man enters and dances with the girls. The man with the "blue" t-shirt represents Klenow polymerase, a mesophilic protein. Klenow is the "large fragment" of DNA polymerase I from Escherichia coli (37°C). The right leg of the man is the active "proofreading" domain of Klenow. Klenow can differentiate pt-DNA from ds-DNA. The man interacts more with the girl on the left side than with the girl on the right side, illustrating that Klenow binds pt-DNA tighter than ds-DNA.

4. Part II: Klentaq-DNA Interaction. Klenow and Klentaq are considered functional homologues due to their sequence and structural similarities. Since Klenow and Klentaq "look" similar, the man who plays Klenow in Part I also represents Klentaq in Part II. As Klentaq polymerase, a thermophilic protein, the man wears a "red" t-shirt. Klentaq is the "large fragment" of DNA polymerase I from Thermus aquaticus (75°C). Unlike Klenow, Klentaq does not have an active "proofreading" domain. Klentaq cannot differentiate pt-DNA from ds-DNA. The man interacts similarly with the girls, illustrating that Klentaq binds pt-DNA and ds-DNA with similar affinities.

5. Part III: Fluorescence Anisotropy. This technique was one of the methods used to measure the interactions of protein and DNA in this study. Fluorescence anisotropy is a technique that measures the tumbling rate of molecules. A DNA molecule is smaller than a protein-DNA complex, so DNA can move faster than protein-DNA complex. After proteins are added and these proteins bind to the DNA molecules, the protein-DNA complexes move more slowly. After the DNA is saturated with protein, there is no more change in the anisotropy values. That ends the experiment and the dance. (Trivia question: Can you spot the fluorophore on the DNA?)

Science and Art make the People come together!!! (inspired by the song "Music" by Madonna).

Thank you for watching!!!

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