Genetics:
Protein Reverses Chromatin Engineering
Biological Chemistry:
Molecule Implicated in Transcription Termination
Structural Biology:
DNA Splicing Enzyme Observed in Action
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Brigham Celebrates 50th Anniversary of First Human Organ Transplant
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Joslin Names Conley Chairman of the Board
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Macklis Receives Javits Neuroscience Investigator Award
Global Citizen Award Goes to Bill Moyers
HMS Family Health Guide Published in Paperback
New Appointments to Full Professor
Honors and Advances
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DNA Splicing Enzyme Observed In Action
Ligase Encircles DNA While Connecting Genetic FragmentsUnder the sink, a plumber joins two pieces of pipe with a circular fitting. Behind the home entertainment system, a video technician splices wires together with a plastic cuff.
Time to make the doughnuts. During chromosome replication, DNA is continuously synthesized on one daughter strand, a process that follows behind the unzipping parent chromosome. On the other daughter strand, the copying machinery moves in the opposite direction, making Okazaki fragments in discontinuous DNA synthesis. In the final stage of connecting the fragments, the ligase wraps around the newly made DNA like a doughnut. Inside the ligase ring, one section binds to the DNA. Another part of the ligase untwists and exposes the ends of the Okazaki fragments. The third domain fuses the ends with the adenosine 5'-monophosphate (AMP) molecular welding torch it carries in for the job. (Image courtesy of John Pascal)
And dividing cells dispatch a fix-it molecule that encircles DNA to join its loose ends in the final stage of chromosome replication, according to the first published crystal structure showing the shape of a DNA ligase at work. (See also companion story.)
The paper, appearing in the Nov. 25 Nature, shows that, huddled around a coil of DNA, human ligase I resembles a doughnut. The enzyme wraps around a length of freshly made chromosome, unwinds it a twist to expose the DNA ends, fuses the ends together, and moves on to repeat its task, said senior author Tom Ellenberger, the Hsien Wu and Daisy Yen Wu professor of biological chemistry and molecular pharmacology at HMS.
The details revealed by the ringlike structure suggest new ideas about the functions of this enzymatic workhorse and its two specialized cousins, ligases III and IV, which tend to occasional repairs of chromosomes dinged by life's damaging exposures.
Disease Consequences
Although the clinical implications may not be realized immediately, ligase malfunction in humans may cause chromosome breaks or other genetic errors that can give rise to cancer. In the one known case involving an inherited mutation of DNA ligase I, a young Irish woman had stunted growth, sun sensitivity, and immunodeficiency with recurring infections. She died at age 19, probably of lymphoma. The differences between human and bacterial ligases also may be a potential avenue for developing selective antibacterial agents, Ellenberger said.
John Pascal (left) and Tom Ellenberger report the first crystal structure of human DNA ligase I in action. (Photo by Steve Gilbert)
Just as a drug can be effectively used for decades without doctors understanding exactly how it works, scientists have used bacterial ligases for years in research to cement the ends of DNA together, without knowing the finer mechanistic details.
The need for the ligase in a living cell arises from the traffic flow required to perfectly reproduce a complete set of 46 chromosomes for virtually every new cell in the human body. The process begins with a chromosome unzipping into two strands at the replication fork. On one strand, a copying machine called DNA polymerase motors closely behind, stitching in a continuous string of complementary nucleotides to make a tidy new double helix.
The other strand can only be copied in the opposite direction. On this template, another polymerase tackles small sections, repeatedly backtracking then jumping ahead while manufacturing a series of DNA segments behind the replication fork. The sections of the newly synthesized strand--Okazaki fragments--are temporarily tagged with RNA primers that are eventually removed by other enzymes. Left unattended, loose
DNA ends are prone to recombining with other DNA in potentially harmful ways. For each new set of chromosomes, that leaves about five million split ends to patch together. "We're talking about a lot of ligation," Ellenberger said.
Freeze Frame
Previously, other researchers solved the crystal structures of solo bacterial and viral ligases, but it was hard to imagine how they might grab the ends of the DNA. Much of science begins with the simplest organisms and works its way up to investigate the more complicated and specialized analogues in humans. This time, the complexity of the human ligase made for a more stable molecular complex with DNA than the simpler viral ligases, which was necessary for the crystal structure, said postdoc John Pascal, first author of the paper.
| "I rarely take work home, but I took this picture home to show my wife what I've been working on for the last 10 years." |
The final structure shows three main components. Unlike the simpler two-domain ligases, human DNA ligase I has an additional section called the DNA-binding domain that serves as an operating table where the DNA is splayed out. The second part of the molecule unfurls the DNA and exposes the loose ends. The third domain catalyzes the joining reaction that seals the gap.
Biologists have known what a ligase does for 40 years, but this is the first time they have seen an action shot. "I rarely take work home, but I took this picture home to show my wife what I've been working on for the last 10 years," said co-author Alan Tomkinson, of the University of Maryland School of Medicine, who stuck it on the refrigerator.
Now, Pascal and Ellenberger are following up to learn how the ligase interacts with the other proteins in the precision drill team of replication. They are especially interested in how the ligase may squeeze against another protein ring left by the polymerase at the end of each segment. This ring, a sliding clamp called the proliferating cell nuclear antigen (PCNA), first tethers the polymerase to the template strand during synthesis of a new chromosome. Once abandoned by the polymerase, it orchestrates the series of enzymes that tend to the final stages of DNA replication and repair, including recruiting the ligase for the final fusion.
--Carol Cruzan Morton