While peering through an electron microscope in 1979 at the University of Oregon, Dr. Andrew Taylor made an observation that left him scratching his head. Taylor had concocted a mixture of DNA and an enzyme that the E. coli bacterium uses to mend broken chromosomes. But instead of the unraveled DNA strands he expected to see, Taylor observed bizarre structures with DNA loops that he could only describe as rabbit ears.
"I immediately called to Gerry that he should come take a look," said Taylor, a staff scientist in Dr. Gerry Smith's Basic Sciences Division laboratory. "I knew that we must have discovered something important, but we didn't know what."
Smith, whose laboratory moved to the center in 1982, added the DNA loops question to a running list of unsolved scientific cases he kept on a blackboard in his office, where it remained for more than two decades.
Now, Taylor and Smith have discovered the solution to their laboratory's Stonehenge-like mystery. In the June 19 issue of Nature, they report that the curious DNA structures represent the tracks of a dual-engine chromosome-repair machine that is different from proteins scientists have seen before. Their analysis of this unusual protein sheds light on how the bacterium tackles one of biology's most important and ubiquitous challenges: the seamless repair of a broken chromosome.
Genetic weaving works
Unless a cell can sew together a broken and frayed chromosome without losing a stitch, vital bits of the genetic blueprint can be lost forever, which can lead to death or diseases such as cancer. For this reason, cells don't have the luxury of simply knotting together broken chromosome ends. Instead, they must use an intricate genetic weaving strategy, known as homologous recombination, in which the torn DNA ends mesh in perfect synchrony with another intact DNA molecule in the cell that acts as template for the repair. If bits of the DNA strands at the broken ends are missing, a part of the homologous recombination system devoted to DNA synthesis fills in the gaps, restoring any lost information.
In addition to its role in DNA repair, homologous recombination also ensures the exchange of genetic information between maternal and paternal chromosomes during meiosis, the process by which eggs and sperm are formed. Such DNA swapping is not only important for generating species variation, it is also essential for the accurate partitioning of chromosomes into the newly created germ cells.
Smith and Taylor study homologous recombination in E. coli, a model organism that has enabled researchers to understand the DNA repair process in greater detail than has been possible in any other organism. The approach has facilitated the analysis of homologous recombination in human cells, in which improperly mended chromosomes are a common feature of many cancer cells. A well-known example is an inherited form of breast cancer that results from defects in the BRCA1 protein, which is important for the homologous DNA repair system in human cells.
Maintaining the proper pattern
"If you want to repair broken DNA with fidelity, there's only one way to do it-by homologous recombination," Smith said. "You could just jam the two ends together-it's better than nothing-but you'll wind up with some DNA that's missing or added. If the wrong ends are joined together, that causes a type of mutation called a translocation, a hallmark of many cancerous cells."
A cell's homologous recombination system must act like a sophisticated sewing machine that can snip, unravel and mend. Because the genetic code is based on the particular ordering of DNA building blocks known as bases (A, C, G and T), restoring the proper meaning to the code is much like aligning or reweaving torn pieces of a plaid or checkered cloth so that the pattern remains in register.
RecBCD's 'twin loops'
The darning process begins with the unwinding of the broken DNA double helix to yield two single strands, one of which will be precisely threaded and matched together with its corresponding partner in the intact DNA template. Most DNA-unwinding enzymes, which are known as helicases, produce a Y-shaped structure similar to a partially unwound piece of licorice, the image Taylor had expected to see under the microscope. Yet the E. coli enzyme, called RecBCD, produces a structure in which one of the two arms of the Y contains a loop with a short tail.
Taylor and Smith discovered that this oddity results from a special property of RecBCD. The enzyme contains an unwinding function powered by dual engines, a feature that presumably enables it to mend DNA over long stretches.
The researchers used an electron microscope to visualize single DNA molecules unwound by the normal enzyme or by enzymes that were defective in one or the other motor. Based on the DNA structures that formed as a result of unwinding by the mutant enzymes, Taylor and Smith deduced that the two motors-each of which fastens to one of the two DNA strands and walks along it-have different horsepower. Because the fast and slow motors are physically attached, RecBCD faces a logistical challenge as it works its way down the chromosome.
Their results suggest that the loop forms ahead of the slow motor as a compensation method for the two engines to remain in sync.
Over time, if the tails get too long, they sometimes come together to form a second loop, yielding the rabbit ear structures, also known as "twin loops," that Taylor first saw long ago.
Benefit to the bacterium
Proteins that "walk" their way down long molecules such as DNA or muscle fibers are often described as molecular motors because they use energy to power their movement. But Taylor said that an enzyme powered by two non-identical motors is virtually unheard of in nature.
"We could find only one other protein described in the literature that contains two unique motors, and RecBCD has additional unusual features not found in that example," he said. Because of its uniqueness, the researchers speculate that RecBCD may offer some benefit to the bacterium.
"During homologous recombination in E. coli and some other types of bacteria, the double helix is unwound into single strands that are very long," Smith said. "But the unwound strands tend to want to stick back together. The loop formed by RecBCD may be a way to keep the strands apart. That would be a simple answer to what seems like a complex problem."
Still, Smith said that scientists are never truly certain of the reasoning that drives the solution to any process in nature-including the formation of the mysterious rabbit ears Taylor saw long ago in E. coli DNA.
"One of my mentors, Salvador Luria, once said, 'How' questions are for molecular biologists. 'Why' questions are for philosophers."