Scientists decipher missing piece of first-responder DNA repair machine

Friday, October 2, 2009

Scientists from the U.S. Department of Energy’s Lawrence Berkeley National Laboratory and the Scripps Research Institute have uncovered the role played by the least-understood part of a first-responder molecule that rushes in to bind and repair breaks in DNA strands, a process that helps people avoid cancer.

Last piece of MRN puzzel falls into place

With this final piece of the puzzle in place, scientists can better understand how the repair mechanism fends off cancer in healthy people, and conversely, how it helps cancer cells resist chemotherapy. This could enable researchers to develop more effective therapies with fewer side effects.

The team deciphered the poorly understood component using innovative x-ray imaging techniques at Berkeley Lab’s Advanced Light Source, which generates intense light for scientific research. They found that it extends from the repair machinery like a flexible arm and grabs molecules that are needed to help the machine zip severed DNA strands back together.

Their work is published in the October 2, 2009 issue of the journal Cell.

“This not only reveals how life works at a fundamental level, but also promises to guide the development of cancer treatments,” says John Tainer of Berkeley Lab’s Life Sciences Division and the Scripps Research Institute in La Jolla, CA. Tainer co-led the research with Paul Russell of the Scripps Research Institute.

The first-responder machine, a protein complex called Mre11-Rad50-Nbs1 (or MRN for short), homes in on the gravest kind of breaks in which both strands of a DNA double helix are cut. It then stops the cell from dividing and launches an error-free DNA repair process called homologous recombination, which replaces defective genes. If unrepaired, double strand breaks can lead to the proliferation of cancer cells.

Unfortunately, MRN’s laser-like focus on DNA repair means that it also mends broken DNA in cancerous cells. This sometimes stymies chemotherapy treatments that kill cancer cells by inducing double strand DNA breaks.

Because of its key roles — good and bad — scientists have painstakingly studied MRN since 1995 to learn how it works in healthy people, how its mutations promote diseases such as cancer, and to possibly disable it during cancer treatment.

Despite more than a decade of effort, a critical part was missing: a protein called Nsb1 that is represented by the ‘N’ in MRN.

To determine Nsb1′s function, the team used an Advanced Light Source beamline called SIBYLS, which yields extremely high-resolution images of the crystal structure of a protein via a technique called x-ray crystallography. The beamline is also equipped with small-angle x-ray scattering, which can determine a protein’s overall architecture in solution, a critical step that approximates how a protein appears in its natural state — such as inside a cell.

The scientists trained these two tools on human and yeast Nsb1 proteins. (DNA repair is so essential to life that many of the molecular machines that perform it have changed little throughout evolution). Importantly, the team studied Nbs1 bound to a partner protein that opens DNA during the first steps of double strand break repair. This enabled them to observe Nsb1 at work.

They found that Nbs1 attaches to the MR protein complex precisely where the protein complex converges on the DNA break. Nsb1 also bends in the middle like an elbow to channel molecules to the repair site.

These insights offer the best glimpse yet of how Nsb1 works and how damaged Nsb1 can lead to disease. It also suggests ways to monkey wrench MRN so that it can’t repair DNA during chemotherapy. Perhaps a molecule can be wedged into Nsb1′s elbow joint so it can’t bend, rendering the MRN complex useless.

“These crystal and solution structures have given us an exciting leap forward in our understanding of the Nbs1 and how defects in the protein cause disease,” says Scott Classen of Berkeley Lab’s Physical Biosciences Division.

Adds Tainer, “Understanding how the body responds to DNA breaks is fundamental for cancer interventions and gene therapies. These results open the door to controlling the repair of DNA breaks for cancer therapeutics and gene targeting.”

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DOE/Lawrence Berkeley National Laboratory: http://www.lbl.gov

Thanks to DOE/Lawrence Berkeley National Laboratory for this article.

DNAWellnessinfo.com Resource:  http://www.labspaces.net/99978/Scientists_decipher_missing_piece_of_first_responder_DNA_repair_machine

New Hope For Deadly Childhood Bone Cancer: Surprising Discovery Made By Studying ‘Junk DNA’

ScienceDaily (Sep. 1, 2009) — Researchers at Huntsman Cancer Institute (HCI) at the University of Utah have shed new light on Ewing’s sarcoma, an often deadly bone cancer that typically afflicts children and young adults. Their research shows that patients with poor outcomes have tumors with high levels of a protein known as GSTM4, which may suppress the effects of chemotherapy

The research is published online in the journal Oncogene.

“Doctors and researchers have long known that certain Ewing’s sarcoma patients respond to chemotherapy, but others don’t even though they have the same form of cancer,” says HCI Investigator Stephen Lessnick, M.D., Ph.D. “Our research shows that GSTM4 is found in high levels among those patients where chemotherapy doesn’t seem to work. It’s found in low levels in patients where chemotherapy is having a more positive effect.”

The research could lead to drugs that can suppress GSTM4 in certain patients. It also could lead to a screening test that could reveal which therapies will be most effective for patients. “GSTM4 doesn’t seem to suppress the benefits of all chemotherapy drugs, just certain ones. A GSTM4-based test could help to identify the best therapy for each individual patient,” Lessnick says.

Ewing’s sarcoma is the second most common bone cancer in children and adolescents. The five-year survival rate is considered poor at about 30 percent if the cancer has spread by the time it is diagnosed, and there is an even poorer prognosis for patients who have suffered a relapse.

For this study, researchers focused on an abnormal protein known as EWS-FLI, which is found in most Ewing’s sarcoma tumors. What they discovered is that EWS-FLI causes increased amounts of the GSTM4 gene – and the protein it produces – to be expressed in tumors, a previously unknown effect that led them to make the connection between poor outcomes and high levels of GSTM4. The discovery was made by focusing on repetitive DNA sequences called microsatellites. Microsatellites are sometimes referred to as “junk DNA” because they are not thought to have a normal role in the genome. By examining how EWS-FLI interacts with certain microsatellites, Lessnick and his team were able to identify GSTM4.

Lessnick says the next step in research is to focus on testing and treatments that may lead to better survival rates in patients. “Personalized medicine is the next frontier in the battle against cancer,” he says. “We now know all cancers are not the same. By focusing on how these proteins are expressed in individual tumors, we may soon be able to offer the treatment that will work best for each patient, and that could lead to higher cure rates,” he says.

Lessnick is director of HCI’s Center for Children’s Cancer Research, and is a Jon and Karen Huntsman Presidential Professor in Cancer Research. This research was supported by funds from the Terri Anna Perine Sarcoma Fund, the Liddy Shriver Sarcoma Initiative, the Sunbeam Foundation, the Huntsman Cancer Foundation, and Alex’s Lemonade Stand Foundation.

Scientists Create Custom 3-dimensional Structures With ‘DNA Origami’

ScienceDaily (June 15, 2009) — By combining the art of origami with nanotechnology, Dana-Farber Cancer Institute researchers have folded sheets of DNA into multilayered objects with dimensions thousands of times smaller than the thickness of a human hair. These tiny structures could be forerunners of custom-made biomedical nanodevices such as “smart” delivery vehicles that would sneak drugs into patients’ cells, where they would dump their cargo on a specific molecular target.

While creation of structures from single layers of DNA has been reported previously, William Shih, PhD, senior author of the study appearing in the May 21 issue of Nature, said the multi-layer process he and his colleagues developed should enable scientists to make customized DNA objects approximating almost any three-dimensional shape. Multilayered objects are more rigid and stable, thus better able to withstand the intracellular environment, which “is chaotic and violent, like being in a hurricane,” Shih said. “We think this is a big advance.”

Shih is a researcher in Dana-Farber’s Cancer Biology program. He is also an assistant professor in the Department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School, and a Core Faculty member of the Wyss Institute for Biologically Inspired Engineering at Harvard.

Masters of the ancient Japanese art of origami make a series of folds in a single piece of paper to form stunningly intricate models of animals and other shapes. “We focus on doing this with DNA,” explained Shih. While DNA is best known as the stuff of which genes are made, here the scientists use long DNA molecules strictly as a building component, not a blueprint for making proteins. Shih and his colleagues reported in the Nature paper that they were able to construct a number of DNA objects, including a genie bottle, two kinds of crosses, a square nut, and a railed bridge.

DNA origami is an outgrowth of research in nanotechnology – using atoms and molecules as building blocks for new devices that can be deployed in medicine, electronics, and other fields. Scientists envision using the minuscule structures — which are about the size of small viruses — to mimic some of the “machines” within cells that carry out essential functions, like forming containers for molecular cargos and transporting them from one place to another.

“This is something that nature is very good at — making many complex machines with great control. Nature optimizes cellular technology through millions of years of evolution; we don’t have that much time, so we need to come up with other design approaches,” Shih said.

DNA origami are built as a sheet of parallel double-helices, each consisting of two intertwined strands made up of units called nucleotides. Long strands of DNA serving as a “scaffold” are folded back and forth by short strands of DNA serving as “staples” that knit together segments of the scaffold. The DNA sheet, which Shih likens to the thin bamboo mat that sushi chefs use to prepare maki rolls with filling, is then programmed to curl on itself into a series of layers that are locked in place by staples that traverse multiple layers.

With the design in hand, the scientists then order the DNA staple strands from a company, which take about three days to be synthesized and shipped. Fabricating the desired structure involves mixing the DNA scaffold and staple strands, quickly heating the mixture, and then slowly cooling the sample. This process coaxes the DNA to “self-assemble” and make billions of copies of the desired object. The process takes about a week, though the researchers intend to improve this rate. Finally, the researchers can check the finished product using an electron microscope.

The tiny machines the researchers are aiming for could, for example, act as navigation aids to guide bubble-like sacs filled with medicines. “These machines could be placed on the outside of the drug-delivery vehicles to help them cross biological barriers, or help them outwit mechanisms that are trying to remove things from the bloodstream, so they can reach their target,” suggested Shih.

The technology could also be useful in diagnostics of the future. While current lab tests can measure the concentration of different substances in the body, it may be possible with DNA “to measure the concentration of something within a single cell,” said Shih.

In addition to Shih and Douglas, the authors of the Nature paper include Hendrik Dietz, PhD, Tim Liedl, PhD, Björn Högberg, PhD, and Franziska Graf, of Dana-Farber and Harvard Medical School.

The research was supported by grants from the National Institutes of Health, the Claudia Adams Barr Program, the Wyss Institute for Biologically Inspired Engineering at Harvard, and several fellowships.

1. Shawn M. Douglas, Hendrik Dietz, Tim Liedl, Björn Högberg, Franziska Graf & William M. Shih. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature, 2009; 459 (7245): 414 DOI: 10.1038/nature08016

Marijuana Damages DNA and May Cause Cancer

By LiveScience Staff

posted: 13 June 2009 11:55 am ET

A lot of studies have shown marijuana is not good for you. It can fry the brain and contribute to psychosis. The latest one finds “convincing evidence” that marijuana smoke damages the genetic material DNA in ways that could increase the risk of cancer.

Toxic substances in tobacco smoke can damage DNA and increase the risk of lung and other cancers. However, there has been uncertainty over whether marijuana smoke has the same effect.

Scientists are especially concerned about the toxicity of acetaldehyde, present in both tobacco and marijuana. However, it has been difficult to measure DNA damage from acetaldehyde with conventional tests.

Using a highly sensitive new method called modified mass spectrometry, Rajinder Singh at the University of Leicester and colleagues found the data they sought, they report in the June 15 issue of Chemical Research in Toxicology, a journal of the American Chemical Society.

“These results provide evidence for the DNA damaging potential of cannabis [marijuana] smoke, implying that the consumption of cannabis cigarettes may be detrimental to human health with the possibility to initiate cancer development,” the researchers write. “The data obtained from this study suggesting the DNA damaging potential of cannabis smoke highlight the need for stringent regulation of the consumption of cannabis cigarettes, thus limiting the development of adverse health effects such as cancer.”

Earlier this year, a separate study found evidence that adolescents and young adults who smoked a lot of marijuana are more likely than non-users to have disrupted brain development. Research in 2007 found pot smokers have on average a 41 percent increased risk of developing psychotic disorders later in life.

The study was funded by the European Union Network of Excellence, the Medical Research Council and other groups.

DNAWellnessinfo.com Resource:  http://www.livescience.com/health/090613-marijuana-dna-cancer.html