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On the Road to Discovery
CHORI Scientist Elucidates Structure and Mechanism of the Bacillus stearothermophilus RecU Holliday Junction Resolvase

"We knew the enzyme worked on DNA, so we wanted to somehow look at this process. We didn't have an easy way to do this, so we actually took a structure of Holliday junction DNA and modeled this with our structure."

The crystal structure of RecU Holiday junction resolvase, the protein responsible for DNA repair and recombination in B. stearothermophilus bacteria, has just been reported by the group of Mark Jedrzejas, PhD, in CHORI’s Center for Immunobiology and Vaccine Development. This new high resolution crystal structure provided enough detail to reveal the critical location of two ions, which led to the ability to discover the protein’s entire mechanistic function.

“The earlier data didn’t have a scientific basis for how it worked,” says Dr. Jedrzejas. “We obtained this crystal structure, and from there were able to identify critical ions and model a complex with DNA substrate that shows how this protein actually works.”

The critical ions that provided the key to understanding were two magnesium ions on what Dr. Jedrzejas refers to as a mushroom-like structure with a positively charged stalk that appeared to be designed to fit exactly into mobile, four-strand, DNA structures called Holliday junction DNA.

Using the charge distribution on the surface of the protein as reference, Dr. Jedrzejas and his colleagues were able to fit together the RecU and Holliday junction DNA to obtain a complex.

"The moment we modeled the complex, we knew we were home," Dr. Jedrzejas says.

This was due to the location in the model of two water molecules and how they coordinated with the two previously elusive magnesium ions as well as to significant amino acid residues necessary for catalysis. All of these players were positioned in the model exactly where they needed to be in order to cleave DNA, the catalysis for which RecU is responsible.

"The model is consistent with one of the water molecules acting as a nucleophile and the other as a general acid," explains Dr. Jedrzejas. "The catalytic amino acid residues act as the general base, the general acid, and the Lewis acid. Structural modifications, or dynamic behavior, of both the protein and DNA are essential for catalysis."

Knowing how the molecule functions is the first step to understanding potential ways of interfering with bacterial functionality involved in its reproduction.
"You always want to understand how bacterial organisms work, because from this understanding, you might deduce how that information could be applied in a particular situation to design a cure or treatment of some sort."
Once you know the molecular mechanisms," says Dr. Jedzrejas, "then you have the tools to make the mechanism better or worse to some desired effect."

In this case, much more study of the protein is required. What is known is that RecU is involved in DNA repair and recombination, but what might occur as a result of this action is largely only theory.

"If bacterial DNA contained an error, for example, the bacteria would want it repaired, to prevent the error from being multiplied and carried on, potentially rendering the bacteria ineffective. Recombination on the other hand might facilitate evolutionary processes to make bacteria better through DNA modifications," suggests Dr. Jedrzejas.

"The protein itself will need to be studied more carefully. What we have provided is the molecular mechanism of action, the key to how the molecule actually works."

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Monday, May 16, 2011 11:33 PM

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