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Biochemists uphold law of physics
Date:7/15/2013

In 2001, Kowalczykowski's laboratory, with the late professor Ronald Baskin at UC Davis, developed a technique to trap single molecules of RecBCD and watch them at work on a strand of DNA in real time. They have since exploited the method to study how DNA is repaired -- in humans, a vital process in protecting against cancer and developmental defects.

"Ever since the original experiments, we've noticed RecBCD molecules have quite a broad range of speeds," Kowalczykowski said.

Liu used the single-molecule visualization technique to measure the rates of hundreds of RecBCD molecules, finding bell-shaped curves for the whole population.

One explanation could be that a large proportion of the proteins were not folded properly and were "trapped" in an inefficient state. However, mild heat or unfolding treatments, which should have allowed the proteins to relax into their correct folded state, had no effect.

RecBCD usually runs for about a minute before stopping spontaneously. Liu found that he could stop the enzyme early by taking away ATP, the chemical fuel that makes the enzyme work.

When he brought back the fuel, he found that the enzymes started up again -- but at a random speed, not related to their previous rate. Overall, the individual RecBCD proteins could restart at any speed within the bell-shaped spread shown by all the proteins.

The experiment shows that RecBCD can move through a wide range of slightly different conformations in which it works at slightly different speeds. However, when it is attached to a step on the DNA ladder, it is locked in shape. Because the time for the enzyme to move from step to step along DNA is shorter than the time it needs to change conformation (about one second), it remains in the same conformation as long as it is moving along DNA, Kowalczykowski said.

What is the point? Why not just have all the enzymes work at one, optimal rate? Having this important enzyme
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Contact: Andy Fell
ahfell@ucdavis.edu
530-752-4533
University of California - Davis
Source:Eurekalert

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