Boston, MA (October 12, 1999) -- Harvard researchers have created the first atomic-resolution image of a donut-shaped enzyme, or helicase, that unwinds the DNA double helix to expose its genetic letters for DNA replication. Michael Sawaya, postdoctoral fellow in the lab of Tom Ellenberger, associate professor of biological chemistry and molecular pharmacology (BCMP), 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.
"We know next to nothing about how these helicases move on DNA," says Ellenberger. 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. Helicases are interesting for several 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, ploughing 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 came 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.
For Ellenberger, the helicase represents a step in his ultimate goal to crystallize the entire replication fork of the bacteriophage T7, a complex of five different types of protein that copies DNA. Last year, researchers led by Ellenberger and Charles Richardson, professor of biochemistry and molecular pharmacology, reported the crystal structure of T7's DNA polymerase. 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 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 donut sits atop a smaller one that is the primase, another enzyme of the replication fork. 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 donut'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, or conformation 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, 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 donut'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, in that 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."
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