After a decade-long struggle, researchers have determined the structure of an enzyme that repairs damage wreaked by the sun on DNA and has an important role in preventing skin cancer.
Ultraviolet light from the sun can cause DNA damage by fusing together two of the nucleotide bases that sit side by side on a DNA strand. This forms a bulky lesion that distorts the DNA helix, making it impossible for most of the enzymes involved in DNA replication to read past the altered site correctly.
In 1999, researchers reported that one enzyme, a DNA polymerase, a member of a family of proteins that copes with DNA damage, is able to bypass this error. The enzyme is mutated in some patients with a condition called xeroderma pigmentosum, which causes extreme sensitivity to sunlight. For such patients, the briefest exposure to the sun can be enough to cause skin cancer. Two research groups report in Nature that they have at last determined precisely how DNA polymerase manages this feat. The teams have captured a molecular snapshot of the enzyme, both from the yeast Saccharomyces cerevisiae and from humans, as it reads damaged DNA and produces a pristine, unmutated copy.
“These two papers represent a major step forward in understanding the basic mechanisms responsible for skin cancer,” says Thomas Kunkel, a biochemist at the National Institute of Environmental Health Sciences in Research Triangle Park, N C, who was not affiliated with either study.
It is an achievement that many labs have been striving for, says Kunkel. “My own lab has been trying to get the answers that are in these papers for at least three years,” he says. “We’ve been pouring a lot of energy into this, but we just couldn’t solve the structures.” The problem lay with the protein crystals that are often used to determine molecular structures, says Satya Prakash, a biochemist at the University of Texas Medical Branch in Galveston and an author on one of the Nature papers. These crystals consist of many copies of the protein assembled together in a repeating matrix. Interactions between the proteins in the crystals freeze the polymerase in place, making it impossible for researchers to observe how it processes DNA. “We spent nine years wandering around, doing a million things,” says Prakash. “Frankly, there were times when I said, ‘You know what? We should just forget it.’” But then Prakash’s collaborator, Aneel Aggarwal of Mount Sinai School of Medicine in New York, thought to mutate the two amino acids in yeast’s DNA polymerase that were interacting with other copies of the protein in a crystal. Meanwhile, Wei Yang at the National Institute of Diabetes and Digestive and Kidney Diseases in Bethesda, Md., and her colleagues took a similar approach using the human version of the protein.
Both teams converged on the same explanation for the unusual abilities of the DNA polymerase. Like other DNA polymerases, the enzyme is shaped like a hand, with distinct finger, palm and thumb regions. The enzyme holds onto DNA like a hand grasping a rope. The finger and palm regions of the protein interact with the DNA and form what is known as the “active site,” where catalysis takes place.
In DNA polymerase, this active site is larger than in other similar DNA-replicating enzymes. The roomier active site is able to accommodate the bulky lesion caused by ultraviolet light, enabling the protein to read two fused thymine bases, insert the correct matching base pairs (adenine, adenine) in a new DNA strand, and proceed on down the chain. Furthermore, the enzyme has a stiff “molecular splint” region that prevents the template DNA from slipping out of its normal conformation. Yang’s team determined that eight of the mutations sometimes found in patients with xeroderma pigmentosum interfered with either the molecular splint or the enlarged active site.
With these structures known, Kunkel says the field is poised to learn more about how cells cope with the damage caused by sunlight and perhaps other factors. “It is almost certainly going to be true,” he says, “that this enzyme has other important functions in the cell besides just bypassing ultraviolet damage.”