Unusual Mechanism Keeps Repair Protein Accurate

Cancer researchers have discovered that a recently identified protein critical for repairing damaged genes uses an unusual mechanism to keep its repairs accurate.

The protein, called DNA polymerase lambda, is one of a group of proteins known as DNA polymerases that are vital for accurately making and repairing DNA.

But while other DNA-repair proteins insure their accuracy with the help of so-called proof-reading regions or accessory molecules, this protein maintains its accuracy using an otherwise ordinary-looking portion of its molecular structure.

The study was led by Zucai Suo, assistant professor of biochemistry and a researcher with the Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute. The research, published in the July 14 issue of The Journal of Biological Chemistry, provides new insights into how cells repair damaged DNA.

“DNA is constantly attacked and damaged by a variety of agents,” Suo says. “The body must properly repair that damage, or it can lead to cell death or to cancer, birth defects and other diseases.

“There are six families of DNA polymerases,” Suo says, “and this is the first polymerase to use this mechanism to maintain its accuracy when making new DNA. It is both surprising and unprecedented.”

The repair protein itself was first discovered by scientists studying DNA sequence data produced by the Human Genome Project. Suo and his colleagues then became interested in learning how the repair protein worked.

The protein has four distinct regions, or domains. Three of the regions had molecular structures that strongly suggest the task they performed.

For example, regions three and four closely resemble a well-known repair protein called DNA polymerase beta. In fact, it was this similarity that tipped off scientists that the new protein was probably involved in DNA repair.

Region one also had a predicted structure that should allow it to “dock” with other proteins. “This suggests that this protein may do more than just fix DNA damage,” Suo says.

Region two held the surprise. It is called the proline-rich domain because it has high levels of the amino acid proline.

“There was no known function for a structure like the proline-rich domain, so we at first thought it did nothing more than connect the docking region of the protein with regions three and four,” Suo says.

“Then by accident we learned that this was not just a structural connection, but that it is critical to the protein's ability to replicate DNA with very few mistakes.”

For this study, Suo and his colleagues wanted to learn how efficiently the new protein made new DNA. But the researchers initially considered the protein too large and difficult to produce in the laboratory. So instead of making the entire protein, the researchers made only the part that does the repair work, regions three and four.

When they tested this short version of the protein, however, they found that it made up to a 100 times more mistakes than did the similar repair protein, DNA polymerase beta.

“That error rate is too high,” Suo says. “If the entire repair protein produced that many errors, it would cause more problems than it would fix.”

Next, the researchers made the entire protein and found that it could repair DNA as accurately as the comparison protein.

Last, they tested a version of the protein that lacked the docking region. This shortened molecule also accurately made DNA.

“To find that the proline-rich domain was responsible for this repair protein's high fidelity came as a complete surprise,” Suo says.

Presently the scientists are studying the three-dimensional structure of the entire protein to learn how the presence of a proline-rich region influences the ability of the protein to accurately make DNA.

Funding from the National Institutes of Health Chemistry and Biology Interface Program and from the American Heart Association Predoctoral Fellowship program supported this research.

Sources:
Ohio State University
Science Daily

Old as your genes

A fingerprint of gene activity could reveal the true 'youthfulness' of our kidneys, hearts and muscle, regardless of our biological age. The technique might one day be used to find healthy organs for transplants or to warn us of impending disease.

It's hard to tell, particularly on a cellular level, whether a young and healthy body conceals a withering heart — or conversely, whether an old man has a vigorous ticker like that of a younger man.

Stuart Kim of Stanford University Medical Center, California, says that a simple genetic test might do the trick. He and his colleagues have found a set of genes whose activity reveals how well organs are operating, regardless of their owner's actual age.

The team analysed the activity of thousands of genes in 81 muscle samples from people aged between 16 and 89. They pulled out a set of 250 genes whose activity goes markedly up or down with age.

When they compared the activity of these genes with the muscle fitness of individuals, measured by the size of their muscle fibers, they found that the genetic profile, rather than a person's age in years, was a more accurate indicator of fitness.

Body clock

The speed with which our cells and bodies deteriorate is determined partly by the genes we inherit from our parents and partly by the ravages of living. These factors can change the rate at which certain genes manufacture proteins, and other aspects of the cell's machinery. Some studies, for example, have shown that the ends of chromosomes, called telomeres, decay over time and do so faster in those who indulge in unhealthy activities such as smoking.

Kim's team found one 64-year-old man who had a pattern of gene activity more like that of a younger person. Indeed, under the microscope, his muscle appeared young; it contained bigger 'fast twitch' fibers that are good for sprinting and more prevalent in young muscle. The findings are reported in the journal PLoS Genetics1.

In an earlier study, the same group detected a 78-year-old woman with a kidney more like that of a centenarian, according to her genetic profile and an inspection of the tissue under the microscope2.

The researchers found that aging affects some of the same genes across many different tissue types and in many different animals. One group of genes, which is involved in generating energy in the cell's mitochondria, quiet down with age in human muscle, kidney and brain tissue, and also in aging mice and flies, even though these animals have very different lifespans.

It may be that this pathway is a weak spot in the cell that is particularly vulnerable to aging, Kim says.

Healthy living

Kim says that such techniques could one day be used to identify donor organs that are normally ruled out because of the donor's age but may actually be in good working order. "We could open up a huge new pool of donors," Kim says, who is planning a study to test this idea.

In future, a routine blood test at the doctor's office could also reveal the true working condition of organs, allowing patients to modify their lifestyle or diet to rejuvenate their bodies.

But to do this researchers will need to find a way to gauge the activity of an organ's genes from molecules in the blood rather than from a tissue sample, which is difficult to obtain.

Source:
news @ nature.com

Neanderthal genome

We have the modern human genome. Now researchers are set to sequence the DNA of our extinct cousins: Neanderthal man.

The Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, in collaboration with 454 Life Sciences Corporation, in Branford, Connecticut, today announce a plan to have a first draft of the Homo neanderthalensis genome within two years.

Comparing the result to modern human and other primate genomes should help to clarify the evolutionary relationship between humans and Neanderthals. It may also illuminate the genetic changes that enabled humans to leave Africa and rapidly spread around the world around 100,000 years ago.

The chimpanzee (Pan troglodytes) has already been sequenced and stands ready to be compared to Neanderthals (see Chimp genome special). The US National Human Genome Research Institute (NHGRI) has set a goal of sequencing the genome of at least one genome from each of the major positions along the evolutionary primate tree, including the rhesus macaque, orangutan, marmoset, northern white-cheeked gibbon and gorilla.

DNA hunt

The announcement comes as scientists gather in Bonn, Germany, this week to mark the 150th anniversary of the discovery of Neanderthal man — made in Germany's Neander Valley. During 21-26 July, experts will debate all aspects of Neanderthal life, from how they migrated across Europe to what effect climate may have had on their evolution. They will also debate how to find more and better samples to work with (see Palaeoanthropology: Decoding our cousins).

Getting clean genetic material out of such ancient bones is a challenging task. The DNA of the bacteria and fungi that degrade a body after it dies tends to get mixed up with the DNA of the host. And what hominin DNA does survive is usually broken up into small bits over time.

But there are ways to reduce these problems — including using skeletons left from cannibalistic societies, where no flesh was left on the bones for bacteria to eat.

"The dream to find Neanderthal DNA started in the early 1980s," says Paabo. "The problems with contamination were difficult; I almost gave up at times. But now we have new technologies and fossils free of contamination."

The Leipzig team has already sequenced about one million base pairs of nuclear Neanderthal DNA from a 38,000-year-old Croatian fossil. That success was reported by Svante Pääbo, director of the Institute's department of evolutionary genetics, at a meeting at Cold Spring Harbor Laboratory in New York this May. But they have a long way to go; the entire genome is thought to be 3 billion letters long.

Mother's own

In addition to Pääbo's work with the Croatian fossil, there have been successes with mitochondrial DNA — a portion of the genome that tends to be better preserved, but which makes up only a tiny fraction of the entire sequence and is passed down only through the female line.

Almost ten years ago, Pääbo succeeded in sequencing Neanderthal mitochondrial DNA. More recently, such DNA was extracted from a 100,000-year-old Neanderthal fossil found in Belgium.

But a map of the nuclear DNA will prove the real prize, revealing much more about the Neanderthal genetic make-up.

The project will extract the nuclear DNA from bones or teeth from both the first Neanderthal specimen ever discovered, and some additional bones found in Croatia.

Source:
news @ nature.com

Mapping the protein world

A software package called ARP/wARP is helping to expose the hidden world of biological molecules.

In the early days of X-ray crystallography obtaining a three-dimensional model of a protein required wire models, screws, bolts and years of tedious calculations by hand. Today macromolecular models are built by computers – thanks to sophisticated software and in particular a package called ARP/wARP. Developed by Victor Lamzin at the Hamburg Outstation of the European Molecular Biology Laboratory (EMBL) and Anastassis Perrakis at the Netherlands Cancer Institute (NKI) in Amsterdam, ARP/wARP is currently used by over 2,000 researchers throughout the world. The capabilities of this software will now expand even further - thanks to a grant of over 800,000 US Dollars from the U.S. National Institutes of Health (NIH).

The grant, which will run over four years, comes at a perfect time. "More than 1,000 research laboratories from over 50 countries are holding ARP/wARP licenses and by June this year our paper that described the key innovative feature of ARP/wARP in Nature Structural Biology in 1999 has reached the magic number of 1,000 citations in the scientific literature. This has created an incredible drive for further scientific development," Lamzin says. "The new funding gives us a push to advance the software's ability to recognise and distinguish different types of macromolecular objects, for example DNA, and to improve the automated generation of structural models. ARP/wARP has made the life of structural biologists worldwide a lot easier and will do even more so once the new features planned under the NIH grant have been implemented."

ARP/wARP transforms 'electron density maps', produced in experiments that bombard protein crystals with X-rays, into 3-dimensional structures. "X-ray experiments result in 'diffraction patterns' that can't be interpreted using our eyes," Lamzin says. "These have to be reconstructed into a three-dimensional image through mathematics and models. This was a very tedious, time-consuming, and subjective process."

ARP/wARP was the first, and for a while the only, software that could generate models to fit experimental data automatically and very accurately. It has cut down the time necessary to create structural models from weeks to minutes.

This grant will allow the scientists to explore new concepts of model-building and enlarge the scope of data that the software can handle. ARP/wARP deals very well with high-resolution data that allows to distinguish individual atoms, but much of the data that scientists have to deal with is of lower quality. The software has steadily been improved to work with lower-resolution data, and Lamzin and Perrakis know how to stretch it even further.

"The high-throughput revolution in Structural Biology allows us to work on more and more complex problems relevant to human health," Perrakis says. "Knowing the structures of molecules that play crucial roles in cancer, cardiovascular and neurodegenerative diseases and molecules from pathogenic bacteria or viruses will contribute to design new revolutionary therapeutic strategies."

To meet this objective the scientists intend to study crystals of proteins bound to diverse drug candidates or containing different types of large molecules.

"ARP/wARP needs to meet a two fold challenge: firstly, it needs to be able to work with structural information at lower resolution, within the range of 3.0 to 3.5 Ångstroms, and secondly, the models produced have to be complete and validated. The new NIH grant will help us to approach these aims. In the future researchers will be able to focus on structure analysis rather than just building the structure and, who knows, by combining ARP/wARP with new cell imaging techniques we might be able to model the molecules of a complete cell," Perrakis concludes.

Contact: Anna-Lynn Wegener
[email protected]
0049-622-138-7452
European Molecular Biology Laboratory

Sources:
EurekAlert

Cancer antibody strategy

Researchers at The Scripps Research Institute and The Skaggs Institute for Chemical Biology have developed a unique assembly strategy to produce an anticancer targeting antibody, an approach that combines the merits of small molecule drug design with immunotherapy.

Among the potential therapeutic advantages of this approach is a dramatically increased circulatory half-life of the compound, which could give patients greater exposure to the benefits of any treatment.

In a study, the scientists created what is known as a 'chemically programmed antibody' by using small cell-targeting molecules and a non-targeting catalytic monoclonal aldolase antibody in a novel self-assembly strategy. This compound was evaluated in the treatment of metastatic breast cancer.

The new study achieved a significant enhancement of the treatment of metastatic breast cancer in animal models. The study showed the new hybrid compound remained in circulation for a week. In comparison, the small molecule drug was cleared in a matter of minutes.

"Although the study focused specifically on breast cancer, these new findings could have broad application in the treatment of a number of other cancers, potentially increasing the efficacy of a number of existing or undeveloped small molecule therapies," said Dr Subhash Sinha, associate professor in the Scripps Research department of molecular biology and the Skaggs Institute for Chemical Biology.

Until recently, it had been widely accepted that while antibodies possess a number of therapeutically advantageous traits, treatment with monoclonals required a different antibody for each specific target. However, the scientists have been showing that different small molecule targeting agents - called programming agents or adapters - can be used to selectively direct the same antibody to different sites for different uses so that only a single antibody is required for multiple tasks.

Source:
Pharmaceutical Business Review Online
Scicentists unveil cancer antibody strategy
Helen Marshall