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BIOLOGICAL CHEMISTRY Blueprint Made of Machine That Splits DNAX-ray Crystallography Gives Scientists New Understanding of Molecular Motor Harvard researchers have created the first atomic-resolution image of a doughnut-shaped enzyme that unwinds the DNA double helix to expose its genetic letters for DNA replication. This crystal structure shows six individual helicase proteins assembled into a ring-shaped motor that unwinds the DNA helix. The motor threads one strand through its central hole and forces its way through the base pairs of the DNA double strand. The red balls nestled between two lobes represent dTTP, the motor's fuel molecule, and the pink curl shows the area where the motor grabs onto the DNA. Michael Sawaya, postdoctoral fellow in the lab of Tom Ellenberger, associate professor of biological chemistry and molecular pharmacology, worked out the X-ray crystallographic structure reported in the October 15 Cell. The structure is rendered in pictures that show how six individual polypeptide lobes arrange themselves in space to look a bit like a ring of bread buns. It affords researchers the first detailed glance at a family of proteins that remain enigmatic in spite of their recognized status as a fundamental molecular machine of the cell. In the bacteriophage T7, five different proteins collaborate to replicate its DNA. Tom Ellenberger hopes, someday, to crystallize the whole shebang. For now he published the crystal structure of the helicase half of the helicase–primase protein that spearheads this complex by splaying the DNA strands. The helicase is the larger half of the protein double-decker, depicted here in its silhouette obtained by electron microscopy. This low-resolution image, supplied by Edward Egelman of the University of Minnesota Medical School, guides Michael Sawaya in his ongoing project to determine the crystal structure of the helicase–primase. Below is the helicase's crystal structure placed into the contours generated by an electronic slice through the lower "bagel." "We know next to nothing about how these helicases move on DNA," says Ellenberger. His basic research on the hexameric helicase of the bacteriophage T7 aims to establish the fundamental mechanisms of this class of enzymes. T7 is simple enough so researchers can study the precise interactions of its protein assemblies, and many of its basic properties may apply to higher organisms, as well. Helicases have become a competitive field of inquiry because defects in human forms underlie several diseases, including the cancer-prone Bloom's syndrome and a disease of premature aging called Werner syndrome (see sidebar). Helicases are interesting for a couple of reasons. First, they are molecular motors, just like myosin, which moves along actin fibrils to contract a muscle, or kinesin, which transports cargo along microtubules. The ring-shaped kind of helicase threads one strand of DNA through its central hole and zips along the double strand at breakneck speed, plowing through 300 paired nucleotides per second while shoving the second strand out of the way. The enzyme is powerful, too. Other researchers have placed "roadblocks" in the helicase's way by binding proteins on the DNA's back. Yet the helicase knocked these off as it forced its way through. Second, helicases come in different shapes—some are monomers, others dimers—and they do all sorts of things. The ring-shaped, hexameric helicase studied here spearheads a complex of enzymes as it pries apart the DNA strands for replication. Other helicases help with DNA repair, recombination, transcription, and more. They probably act wherever DNA needs to open up temporarily. Sequencing data suggests there are hundreds of different helicases in the human genome; even the humble yeast boasts about 50 kinds. The Big PictureFor Ellenberger, the helicase represents a step in his ultimate goal to crystallize the entire replication fork of T7, a complex of five different types of protein that copies DNA (see image). Last year, researchers led by Ellenberger and Charles Richardson, the Edward S. Wood professor of biological chemistry and molecular pharmacology, reported the crystal structure of T7's DNA polymerase (Focus, January 23, 1998). Using biochemistry, Richardson's group had learned earlier that the helicase and the other proteins in the replication fork physically touch each other. Collectively, these interactions make the system work in still mysterious ways. The scientists' big-picture goal, literally, is to solve this question by visualizing the enzymes individually and then putting them back together. Richardson is one of the co-authors on the current paper.
The crystal structure of the helicase does not, actually, represent the way this enzyme occurs in real life. In T7, the helicase is a double-decker protein with two enzyme activities. The large helicase doughnut sits atop a smaller one that is the primase, another enzyme of the replication fork. (But that is a future story. Sawaya began determining the structure of the complete helicase–primase two years ago and is still struggling with its complexity.) Crystal ClearMeanwhile, Richardson's lab prepared a helicase fragment of the helicase–primase that was amenable to X-ray crystallography. Curiously, this fragment crystallized as an open ring, like a lock washer, whereas the biologically active form of the helicase more closely resembles a flat washer. "We think, however, that all interactions we are describing closely approximate what we would see in a closed ring," says Ellenberger.So what did the scientists see? All amino acid sites known to be conserved across helicases of this family from different species turned out to reside near the surface of the doughnut's hole, where one DNA single strand passes through. More importantly, though, the structure gives the researchers a first stab at solving the mechanism of how this motor generates motion from energy, like any engine does. Scientists knew that the helicase splits a phosphate off dTTP—a relative of the fuel molecule ATP—to free up chemical energy. It must somehow convert this into the physical force needed to separate the Watson–Crick bonds joining the double strand DNA base pairs. It also must harness energy for large changes in its shape that allow it to step along the DNA. Finally, it needs a mechanism for grabbing and letting go of DNA. The task lies in understanding precisely how the helicase's six subunits cooperate to make these things happen, and the crystal structure shows a plausible way, says Ellenberger. One dTTP is nestled in the cleft between every two subunits (see image), meaning that changes resulting from every dTTP reaction could spread to two subunits. The dTTP binding site also abuts the DNA binding site at the doughnut's inner ring. When crystallized without dTTP, these DNA binding sites were disordered, and consequently failed to appear as crisp patterns in the crystal structure. Yet when the researchers immersed the helicase crystals in a dTTP solution to allow the dTTP to seep into place, they found that not only was the dTTP sitting in its binding pocket but the adjoining DNA-binding region of the helicase also suddenly became visible. This suggests that the dTTP reaction might be coupled to DNA binding, since dTTP cleavage enables the DNA binding region to "shape up" and grab the DNA. A fraction of a second later, this sequence could occur in the adjacent subunits, and so on throughout the ring. Other researchers have advanced two major mechanistic models for simpler helicases that are now being hotly debated. Sawaya's structure cannot pick a winner largely because it is symmetrical. It does not visualize the larger conformational changes that must occur within the ring as it advances on the DNA. The structure does, however, nurture an old passion, says Ellenberger. "As a kid I always loved engines, and I am still fascinated that you can make a protein function as this large, cooperative assembly to move rapidly down a strand of DNA. You've got all these motions flickering in a nanosecond time-realm. This works only because everything is supremely coordinated." —Gabrielle Strobel DNA Helicase--Ticket to Slow Aging?DNA-unwinding enzymes have been getting plenty of attention ever since researchers elsewhere discovered, three years ago, that a mutation in a human DNA helicase causes a disease that promised scientists a handle on the molecular biology of aging. People with Werner syndrome (WS) age prematurely. After puberty, their hair begins to gray and thin, their skin becomes wrinkled, they develop cataracts and osteoporosis, and generally die in their forties from heart attacks, strokes, or cancer. All of that happens thanks to a single mutation in a single gene. This fact was not lost on David Sinclair. "We became involved with helicases the day the WS gene was cloned," says Sinclair, then a postdoc in the lab of Leonard Guarente at MIT. Progress was quick, and this September, Sinclair assumed his new position as HMS assistant professor of pathology. In a 1997 paper in Science, Sinclair and Guarante described accelerated aging in yeast missing their version of the human WS gene, called SGS1. Four months later, the geneticists reported in Cell why they thought this was so. In short, they found yeast have trouble maintaining certain regions of their genome and that loss of SGS1 makes it worse. As a consequence, stretches of DNA pop out of chromosomes and form circles that replicate and accumulate in the nucleus by the thousands, eventually choking the cells. At HMS, Sinclair will try to find out what this helicase actually does in vivo. "We were pleasantly surprised that, in yeast, the molecular mechanism for aging could be so relatively simple," says Sinclair. Aging will prove more complex in humans, but maybe not overwhelmingly so, despite its manifold symptoms. |
