Structure of DNA Repair Complex Reveals Workings of Powerful Cell Motor

ScienceDaily (Mar. 28, 2011) — Over the last years, two teams of researchers at The Scripps Research Institute have steadily built a model of how a powerful DNA repair complex works. Now, their latest discovery provides revolutionary insights into the way the molecular motor inside the complex functions -- findings they say may have implications for treatment of disorders ranging from cancer to cystic fibrosis.

The new finding solves the long-standing mystery of how a single protein complex known as MRN (Mre11-Rad50-Nbs1, shown here) can repair DNA in a number of different ways.

In a paper published in an Advance Online Edition of Nature Structural and Molecular Biology March 27, 2011, the scientists say that the complex's motor molecule, known as Rad50, is a surprisingly flexible protein that can change shape and even rotate depending on the task at hand.

The finding solves the long-standing mystery of how a single protein complex known as MRN (Mre11-Rad50-Nbs1) can repair DNA in a number of different, and tricky, ways that seem impossible for "standard issue" proteins to do, say team leaders Scripps Research Professor John Tainer, Ph.D., and Scripps Research Professor Paul Russell, Ph.D., who also collaborated with members of the Lawrence Berkeley National Laboratory on the study.

They say the finding also provides a critical insight into the ABC-ATPase superfamily of molecular motors, of which Rad50 is a member.

"Rad50 and its brethren proteins in this superfamily are biology's general motors," said Tainer, "and if we know how they work, we might be able to control biological outcomes when we need to."

For example, knowing that Rad50 changes its contour to perform a function suggests it might be possible to therapeutically target unique elements in that specific conformation. "There could be a new generation of drugs that are designed not against an active site, like most drugs now (an approach that can cause side effects, but against the shape the protein needs to be in to work," Tainer said.

Russell added, "Proteins are often viewed as static, but we are showing the moving parts in this complex. They are dynamic. They move about and change shape when engaging with other molecules."

First Responder

The MRN complex is known as a first-responder molecule that rushes in to repair serious double-strand breaks in the DNA helix -- an event that normally occurs about 10 times a day per cell due to ultraviolet light and radiation damage, etc. If these breaks are not fixed, dangerous chromosomal rearrangements can occur that lead to cancer. Paradoxically, the complex also mends DNA breaks promoted by chemotherapy, protecting cells against cancer treatment.

When MRN senses a break, it activates an alarm telling the cell to shut down division until repairs are made. Then, it binds to ATP (an energy source) and repairs DNA in three different ways, depending on whether two ends of strands need to be joined together or if DNA sequences need to be replicated. "The same complex has to decide the extent of damage and be able to do multiple things," Tainer said. "The mystery was how it can do it all."

To find out, Tainer, head of a structural biology group, and Russell, who leads a yeast genetics laboratory, began collaborating five years ago. With the additional help of team members at Lawrence Berkeley National Laboratory and its Advanced Light Source beamline, called SIBYLS, the collaboration has produced a series of high-resolution images of the crystal structure of parts of all three proteins (rad50, Mre11, and Nbs1), taken from fission yeast and archaea. The scientists also used the lab's X-ray scattering tool to determine the proteins' overall architecture in solution, which approximates how a protein appears in a natural state.

The scientists say that the parts of the complex, when imagined together as a whole unit, resemble an octopus: the head consists of the repair machinery (the Rad50 motor and the Mre11 protein, which is an enzyme that can break bonds between nucleic acids) and the octopus arms are made up of Nbs1 which can grab the molecules needed to help the machinery mend the strands.

In this study, Tainer and Russell were able to produce crystal and X-ray scattering images of parts of where Rad50 and Mre11 touched each other, and what happened when ATP bound to this complex and what it looked like when it didn't.

In these four new structures, they showed that ATP binding allows Rad50 to drastically change its shape. When not bound to ATP, Rad50 is flexible and floppy, but bound to ATP, Rad50 snaps into a ring that presumably closes around DNA in order to repair it.

"We saw a lot of big movement on a molecular scale," said Tainer. "Rad50 is like a rope that can pull. It appears to be a dynamic system of communicating with other molecules, and so we can now see how flexibly linked proteins can alter their physical states to control outcomes in biology."

"We thought ATP allowed Rad50 to change shape, but now we have proof of it and how it works," Russell said. "This is a key part of the MRN puzzle."

An Engine for Many Vehicles

Rad50 and ATP provide the motor and gas for a number of biological machines that operate across species. These machines are linked to a number of disorders, such as cystic fibrosis, which is caused by a defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is a member of the ABC ATPase superfamily.

"Our study suggests that ABC ATPase proteins are used so often in biology because they can flexibly hook up to so many different things and produce a specific biological outcome," Tainer said.

Given this new prototypic understanding of these motors, Tainer and Russell envision a future in which therapies might be designed that target Rad50 when it changes into a shape that promotes a disease. For example, chemotherapy could be coupled with an agent that prevents the MRN complex from repairing DNA damage, promoting death of cancer cells.

"There are some potentially very cool applications to these findings that we are only beginning to think about," Russell said.

The study was funded by the National Cancer Institute, the National Institutes of Health, and the Department of Energy.

Scientists Find a Key to Maintaining Our DNA: Provides New Clues in Quest to Slow Aging

ScienceDaily (Mar. 24, 2011) — DNA contains all of the genetic instructions that make us who we are, and maintaining the integrity of our DNA over the course of a lifetime is a critical, yet complex part of the aging process. In an important, albeit early step forward, scientists have discovered how DNA maintenance is regulated, opening the door to interventions that may enhance the body's natural preservation of genetic information.

Humans have two routes for DNA replication and repair -- a standard route that processes DNA quickly but less accurately, and a high-accuracy route that processes DNA slowly but more accurately.

The new findings may help researchers delay the onset of aging and aging-related diseases by curbing the loss or damage of our genetic makeup, which makes us more susceptible to cancers and neurodegenerative diseases, such as Alzheimer's. Keeping our DNA intact longer into our later years could help eliminate the sickness and suffering that often goes hand-in-hand with old age.

"Our research is in the very early stages, but there is great potential here, with the capacity to change the human experience," said Robert Bambara, Ph.D., chair of the Department of Biochemistry and Biophysics at the University of Rochester Medical Center and leader of the research. "Just the very notion is inspiring."

In the Journal of Biological Chemistry, Bambara and colleagues report that a process called acetylation regulates the maintenance of our DNA. The team has discovered that acetylation determines the degree of fidelity of both DNA replication and repair.

The finding builds on past research, which established that as humans evolved, we created two routes for DNA replication and repair -- a standard route that eliminates some damage and a moderate amount of errors, and an elite route that eliminates the large majority of damage and errors from our DNA.

Only the small portion of our DNA that directs the creation of all the proteins we are made of -- proteins in blood cells, heart cells, liver cells and so on -- takes the elite route, which uses much more energy and so "costs" the body more. The remaining majority of our DNA, which is not responsible for creating proteins, takes the standard route, which requires fewer resources.

But, scientists have never understood what controls which pathway a given piece of DNA would go down. Study authors found, that like a policeman directing traffic at a busy intersection, acetylation directs which proteins take which route, favoring the protection of DNA that creates proteins by shuttling them down the elite, more accurate course.

"If we found a way to improve the protection of DNA that guides protein production, basically boosting what our body already does to eliminate errors, it could help us live longer," said Lata Balakrishnan, Ph.D., postdoctoral research associate at the Medical Center, who helped lead the work. "A medication that would cause a small alteration in this acetylation-based regulatory mechanism might change the average onset of cancers or neurological diseases to well beyond the current human lifespan."

"Clearly, a simple preventative approach would be a key, not to immortality, but to longer, disease-free lives," added Bambara.

DNA replication is an intricate, error-prone process, which takes place when our cells divide and our DNA is duplicated. Duplicate copies of DNA are first made in separate pieces, that later must be joined to create a new, full strand of DNA. The first half of each separate DNA segment usually contains the most errors, while mistakes are less likely to appear in the latter half.

For DNA that travels down the standard route, the first 20 percent of each separate DNA segment is tagged, cut off and removed. This empty space is then backfilled with the latter part -- which is the more accurate section -- of the adjoining piece of DNA as the two segments come together to form a full strand.

In contrast, DNA that travels down the elite route gets special treatment: the first 30 to 40 percent of each separate DNA segment is tagged, removed and backfilled, meaning more mistakes and errors are eliminated before the segments are joined. The end result is a more accurate copy of DNA.

The same situation occurs with the DNA repair process, as the body works to remove damaged pieces of DNA.

Unlike the current work, the majority of aging-related research zeroes in on specific agents that damage our DNA, called reactive oxygen species, and how to reduce them. The new research represents a small piece of the pie, but has the potential to be a very important one.

Bambara's team is investigating the newly identified acetylation regulatory process further to figure out how they might be able to intervene to augment the body's natural safeguarding of important genetic information. They are studying human and yeast cell systems to determine how proteins in cells work together to trigger acetylation, which adds a specific chemical to the proteins involved in DNA replication and repair. Researchers are manipulating cells in various ways, through damage or genetic alterations, to see if these changes activate or influence acetylation in any way.

Though they are far from identifying compounds or existing drugs to test, they do see this research having an impact in the future.

"The translational rate is becoming better and better. Today, the course between initial discovery and drug development is intrinsically faster. I could see having some sort of therapeutic that helps us live longer and healthier lives in 25 years," said Bambara.

The work was funded by the National Institute of General Medical Sciences at the National Institutes of Health. In addition to the researchers at Rochester, Ulrich Hübscher, D.V.M., from the University of Zurich and Judith Campbell, Ph.D., from the California Institute of Technology contributed to the research.