Karolin Luger will spend the next three years seeking a three-dimensional image of how a two-yard-long strand of DNA can be folded into a tiny package inside a cell nucleus.
DNA, the double helix of amino acids that constitutes the blueprint of all life, has to be "naked" at certain points along its microscopic strand in order for proteins to use it to replicate body cells.
"All the information for every living cell is stored in DNA, which is like a long computer tape," Luger said. "The nucleus of every human cell has a two-meter strand of DNA in a compartment one one-thousandth of a millimeter around.
"You can’t just ball the strand up and stuff it in there, because the cell needs access to the information that is stored on the strand at various times."
Luger, an assistant professor of biochemistry and molecular biology at Colorado State University, will pursue her research as one of 15 people in the country named to the 1999 Searle Scholars Program. The Searle program supports the research careers of junior faculty with outstanding potential and will provide $60,000 each year to enable Luger to get her project underway.
Luger, who earned a doctorate in biochemistry and biophysics with honors from the University of Basel in Switzerland, wrote her thesis on how proteins can fold themselves inside cells and will bring this background to her current task.
For example, Luger said when a new liver cell is to be fashioned, the information that is necessary for this process must come from a region perhaps 1.5 meters along the DNA strand. The information needed for the formation of a new corneal cell for the eye might be encoded in another region–say, 1.34 meters along the DNA strand. The question for science is how the proteins known as transcription factors can find the appropriate segments of DNA amid the tangle.
"The crucial point here is that if the wrong regions are read off at the wrong time, the result can be cancer or a developmental disorder," Luger said. "DNA in a living cell is bent and highly distorted, and 98 percent of it exists in that form. Cell nuclei don’t have space for free (i.e., linear) DNA.
"So the question becomes, how do cells determine which areas of the (distorted) DNA get read, and how do they find those regions," she said. "This is comparable to trying to find a specific set of needles in a haystack, in a very defined window of time and in a specific order."
The threads of DNA are spooled around protein clumps and the resulting structures are called nucleosomes. There are four basic scenarios and endless variations theorizing how this wrapped-up DNA is used. Luger prefers one in which DNA and its associated proteins, known collectively as chromatin, partially unwrap, perhaps with help from structural changes in the nucleosome particles.
Luger will investigate whether the transcription factors that actually "read" the DNA. She also will decide which bits of the blueprint are needed at a specific time can bind to very specific sites on the surface of the nucleosomes. The process is complicated because the double-helix structure of DNA bends at sometimes-awkward angles, which would seem to make it difficult for the transcription factors to link chemically to the DNA.
Luger’s first task is to prepare relatively huge (20-thousandths of a gram) amounts of nucleosomes and then form them into regular crystals by removing water from the biological material. This crystalline structure can then be studied by x-ray diffraction, in which x-rays are scattered in particular patterns by electrons in the crystals.
The x-ray irradiation itself requires an extremely powerful x-ray source and will be performed at Argonne National Laboratories near Chicago and at the Advanced Light Source in Berkeley, but many experiments will be performed at Colorado State’s own technology advanced X-ray diffraction facilities. The diffraction patterns are then subject to extensive mathematical manipulation to determine electron densities and enable investigators to interpret the patterns produced. Finally, Luger will begin a long process of matching patterns–by eye and hand–on a powerful graphics computer.
"The pattern recognition (the shapes of the nucleosomes being sought) is slow because we have no information on what fits (on the surface of the nucleosome)," Luger said. "What we do have is experimental data, fraught with errors. It is a very tedious process to arrive at the correct answer.
"At the end of three years, we should know whether we can find a (transcription) factor that binds to the nucleosome," she said. "Ideally, we’ll have a three-dimensional picture of that factor bound to the nucleosome. This might fundamentally change our view of how DNA is used as the blueprint of life in a cell."