Goldberg and his colleagues believed that seeing how Ku binds to DNA might provide answers to some of the questions about the Ku-DNA interaction. The researchers used a technique called x-ray crystallography to visualize the interplay between the Ku heterodimer and DNA. In x-ray crystallography, protein crystals are bombarded with intense x-ray beams. As the x-rays bounce off atoms in the crystal, they leave a diffraction pattern, which can then be analyzed to determine the three-dimensional structure of the protein.
Before they could get a full picture of the Ku-DNA complex, Goldberg and his colleagues decided to study the Ku heterodimer by itself. Their attempts to prepare crystals of the Ku protein yielded only a few usable crystals out of the hundreds they prepared. Fortunately, the scientists were able to use a technique pioneered by HHMI investigator Wayne Hendrickson to solve the complete structure of the Ku heterodimer from a single crystal. The technique, called multiple wavelength anomalous diffraction, was applied during crystallographic analyses performed at the National Synchrotron Light Source at Brookhaven National Laboratory.
After the Ku structure was determined, the scientists moved on to solving the structure of the Ku-DNA complex. In their studies, Goldberg and his colleagues had to mimic DNA breakage, ensuring that their test DNA fragment had only one accessible end -- in order to avoid Ku attaching at more than one site on the DNA. They accomplished this by blocking the other end of the DNA with a bulky DNA motif.
After solving the structure of Ku bound to DNA, Goldberg and his colleagues could see how the Ku heterodimer manages to find a broken DNA end regardless of its sequence. The problem is that Ku is not like a transcription factor that binds to a specific DNA sequence,
Contact: Jim Keeley
Howard Hughes Medical Institute