NEW YORK, Dec. 6, 2006—As biomedical science progresses, physicians apply increasingly refined tools to treat disease. Researchers hope it will eventually be possible to use tools based on the emerging field of nanomedicine. The idea is to repair the body on the tiny scale of molecules—at the nano-scale or roughly one millionth the size of an ant—to reach inside cells and fix what may be broken.
As part of a new National Institutes of Health (NIH) nanomedicine grant, David Roth, M.D., Ph.D., Chairman of the Department of Pathology and the Irene Diamond Professor of Immunology, is collaborating with colleagues at academic research institutions around the country to set up a Nanomedicine Center for Nucleoprotein Machines. The center will be headquartered at the Georgia Institute of Technology, where it will be directed by Georgia Tech biomedical engineer Gang Bao, Ph.D. and molecular biologist William Dynan, Ph.D. of the Medical College of Georgia.
The center’s scientists will focus on the repair of damaged DNA, an essential process that cells perform to preserve the integrity of their genetic material. The first step in the research project is to build tools and do experiments to observe, characterize, and track various stages and types of DNA repair processes.
Just as athletes rely on key muscle groups to power their performance, cells need their protein machines. “Lots of important bodily processes are performed by protein machines that typically contain a number of individual proteins assembled together much like a sophisticated car engine,” explains Dr. Roth. “The machine we elected to study in this project is the DNA repair machine,” he says. “It contains many components and assembles dynamically: its composition also changes with time as different stages of the repair process are completed.”
The team has developed a customized instrument to study a special kind of DNA break.
The miniscule scale at which experiments must be performed is a major challenge. After all, the cell’s nucleoprotein machines—composed of complexes of proteins and nucleic acids—are roughly 100 times smaller than the diameter of a human red blood cell.
To operate at this level, the team will be using a special tool that Bernhard Schnurr, Ph.D. has built. Dr. Schnurr is a post-doctoral fellow in Dr. Roth’s lab, a physicist by training who says he is “amazed by biological systems.” The instrument is a microscope that lets scientists hold and handle a snippet of DNA. Like a yo-yo in water, the DNA segment is suspended in a tiny vial of liquid. The top end is attached to a glass slide and a tiny magnetic bead is affixed to the bottom end.
In this setup, moving a magnet changes the force pulling on the bead, which in turn tugs at the DNA strand. “You change the force by moving the magnet up and down,” says Dr. Schnurr. “If you move the magnet closer to the bead, you pull on it harder and if you rotate the magnets the bead rotates too, much like a compass needle.” The scientists can essentially grip the DNA with magnetic tweezers.
The repair machine
Using these tweezers, the researchers can set up their experiments to observe a special kind of DNA break. Dr. Roth has long been intrigued by a process during which certain cells—precursors to immune cells called lymphocytes—purposefully break their own chromosomal DNA and then repair the breaks. This purposeful break is risky business for the cell. It has even been called “an accident waiting to happen,” says Dr. Roth, since some lymphomas and leukemias may arise when this process is faulty.
At the same time, this process is essential for lymphocyte development, explains Dr. Roth. It helps these cells become a population of superheroes, their bodies studded with special equipment, namely receptor molecules able to recognize and attack millions of invading pathogens.
The cells acquire these qualities in the course of their development in the bone marrow and thymus gland. Not every cell makes it through what appears to be a grueling and selective quality control process. “The thymus and bone marrow are tough schools with a 97 - 99 percent failure rate,” says Dr. Roth. “The mature immune cells that do emerge are stunningly diverse.”
The immune cells owe their diversity to the special way they are made from their genetic blueprint. Following an intentional and specific DNA break, a process called V(D)J recombination occurs. The acronym stands for the V (variable), D (diversity) and J (joining) gene segments that get shuffled around and put together in a variety of combinations. The process is akin to building an architecturally diverse city by taking elements of a blueprint for a single house and recombining them with slight variations. The DNA molecule forms a loop, enzymes cut it at that looped spot, and then hold the loop in place. The gene segments at that location are then combined in many differing ways, explains Dr. Roth.
This recombining step is one of the key events the team wants to study in detail. It relies on a DNA repair pathway known as nonhomologous DNA end-joining that is essential for the repair of many kinds of chromosome breaks and about which little is known.
The proteins that orchestrate the DNA loop formation are recombination-activating gene proteins called RAG1 and RAG2. In their controlled experiment on DNA repair, the scientists plan to watch these proteins, and the subsequent repair of the DNA breaks by nonhomologous DNA end-joining, in action.
Tugging and watching
Using the instrument Dr. Schnurr built, the scientists can flow proteins such as RAG1 and RAG2 into the vial containing the DNA. The microscope is equipped with a camera to record what happens and software that analyzes the observations. The bead and its motion are all the microscope can actually record, explains Dr. Schnurr. The DNA strand is not visible under the microscope. Movements of the DNA strand are inferred from the bead’s motion.
When a loop forms in the DNA, much like a loop in a string, the strand of DNA shortens. Then, if the loop is pulled apart, the strand is back to its original length. By recording how the strand’s length changes, scientists can watch stages of a cellular DNA repair process. “Usually you don’t know where chromosomes are going to break,” says Dr. Roth. In this instance, though, the breaks are not random. The researchers are using proteins with known characteristics—they form loops in the DNA by grabbing it at particular spots and cutting at precise locations. This background knowledge gives the scientists an edge for their experiments. “Given the V(D)J reaction you know exactly where the breaks are going to happen and you can even control when they are going to happen,” says Dr. Roth.
He and his team are looking forward to using this microscope as part of the new nanomedicine venture. It will help them observe and characterize the intricate steps of the DNA repair process as they occur in real time.
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NYU Medical Center
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