DNA Is Dynamic And Has High Energy; Not Stiff Or Static As First Envisioned

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ScienceDaily (July 14, 2009) — The interaction represented produced the famous explanation of the structure of DNA, but the model pictured is a stiff snapshot of idealized DNA. As researchers from Baylor College of Medicine and the University of Houston note in a report that appears online in the journal Nucleic Acids Research, DNA is not a stiff or static. It is dynamic with high energy. It exists naturally in a slightly underwound state and its status changes in waves generated by normal cell functions such as DNA replication, transcription, repair and recombination.

DNA is also accompanied by a cloud of counterions (charged particles that neutralize the genetic material's very negative charge) and, of course, the protein macromolecules that affect DNA activity.
"Many models and experiments have been interpreted with the static model," said Dr. Lynn Zechiedrich, associate professor of molecular virology and microbiology at BCM and a senior author of the report. "But this model does not allow for the fact that DNA in real life is transiently underwound and overwound in its natural state."
DNA appears a perfect spring that can be stretched and then spring back to its original conformation. How far can you stretch it before something happens to the structure and it cannot bounce back? What happens when it is exposed to normal cellular stresses involved in doing its job? That was the problem that Zechiedrich and her colleagues tackled.
Their results also addresses a question posed by another Nobel laureate, the late Dr. Linus Pauling, who asked how the information encoded by the bases could be read if it is sequestered inside the DNA molecular with phosphate molecules on the outside.
It's easy to explain when the cell divides because the double-stranded DNA also divides at the behest of a special enzyme, making its genetic code readily readable.
"Many cellular activities, however, do not involve the separation of the two strands of DNA," said Zechiedrich.
To unravel the problem, former graduate student, Dr. Graham L. Randall, mentored jointly by Zechiedrich and Dr. B. Montgomery Pettitt of UH, simulated 19 independent DNA systems with fixed degrees of underwinding or overwinding, using a special computer analysis started by Petttitt.
They found that when DNA is underwound in the same manner that you might underwind a spring, the forces induce one of two bases – adenine or thymine – to "flip out" of the sequence, thus relieving the stress that the molecule experiences.
"It always happens in the underwound state," said Zechiedrich. "We wanted to know if torsional stress was the force that accounted for the base flipping that others have seen occur, but for which we had no idea where the energy was supplied to do this very big job."
When the base flips out, it relieves the stress on the DNA, which then relaxes the rest of the DNA not involved in the base flipping back to its "perfect spring" state.
When the molecule is overwound, it assumes a "Pauling-like DNA" state in which the DNA turns itself inside out to expose the bases -- much in the way Pauling had predicted.
Zechiedrich and her colleagues theorize that the base flipping, denaturation, and Pauling-like DNA caused by under- and overwinding allows DNA to interact with proteins during processes such as replication, transcription and recombination and allows the code to be read. And back to the idea of the "perfect spring" behavior of the DNA helix - "This notion is entirely wrong," said Zechiedrich. "Underwinding is not equal and opposite to overwinding, as predicted, not by a long shot, that's really a cool result that Graham got."
Support for this work came from the Robert A. Welch Foundation, the National Institutes of Health and the Keck Center for Interdisciplinary Bioscience Training of the Gulf Coast Consortia. The computations were performed in part using the Teragrid and the Molecular Science Computing 85 Facility in the William R. Wiley Environmental Molecular Sciences Laboratory, sponsored by the U.S. Department of Energy's Office of Biological and Environmental Research and located at the Pacific Northwest National Laboratory.
Adapted from materials provided by Baylor College of Medicine, via EurekAlert!, a service of AAAS.

New Drugs Faster From Natural Compounds

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ScienceDaily (July 13, 2009) — Researchers have invented computational tools to decode and rapidly determine whether natural compounds collected in oceans and forests are new—or if these pharmaceutically promising compounds have already been described and are therefore not patentable.

This University of California, San Diego advance will finally enable scientists to rapidly characterize ring-shaped nonribosomal peptides (NRPs)—a class of natural compounds of intense interest due to their potential to yield or inspire new pharmaceuticals.
"These advances will speed the process by which we discover and describe new and biologically active molecules from organisms such as marine cyanobacteria, also known as blue-green algae. This, in turn, will accelerate the timeline for bringing new experimental therapies into clinical application," said William Gerwick, an author on the paper and a professor with the UC San Diego Scripps Institution of Oceanography Center for Marine Biotechnology and Biomedicine and the UCSD Skaggs School of Pharmacy and Pharmaceutical Sciences.
Nonribosomal peptides (NRPs) often serve as chemical defenses for the bacteria that manufacture them. Starting from penicillin, NRPs have an unparalleled track record in pharmacology: most anti-cancer and anti-microbial agents are natural products or their derivatives. However, it is currently difficult, time-consuming and costly to determine the molecular structure of NRPs which, by definition, are not directly inscribed in the genomes of the organisms that produce them.
"NRPs are one of the last bastions of pharmacologically important biological compounds that remain virtually untouched by computational research. As a result, it is currently one of the most painfully slow processes, it is a real bottleneck that we have now removed," said Pavel Pevzner, a computer science professor at UC San Diego's Jacobs School of Engineering and the corresponding author on the Nature Methods paper.
Researchers can now separate known compounds from those that are unknown.
"If I collect 1,000 ocean compounds, why waste time with compounds that are already known or patented?" added Nuno Bandeira, co-lead author on the paper, director of UC San Diego's Center for Computational Mass Spectrometry (CCMS) and a researcher at the UC San Diego division of Calit2, the California Institute of Telecommunications and Information Technology.
"Our algorithms can tell natural product researchers what their compounds are. Manual annotations should be something of the past," said Julio Ng, a co-lead author on the Nature Methods paper and a doctoral student in Bioinformatics at UC San Diego.
"Compound 879," for example, is a cyclic NRP discussed in the Nature Methods paper that was thought to be novel when it was isolated. A lengthy and expensive patenting process, however, uncovered that compound 879 had already been described as an antibiotic and named neoviridogrisen. The new UC San Diego algorithms would have quickly identified this fact. These algorithms make sense of the flood of tiny peptide fragments that are generated by machines called mass spectrometers that blast nonribosomal peptides apart and determine their sizes.
Two complementary processes are used to glean insights from data generated from the mass spectrometers that break the cyclic peptides into smaller and smaller linear pieces.
First, the authors present new algorithms that computers use to piece these peptide fragments back together in order to determine the chemical structure of a cyclic NRP. This is called "De Novo sequencing of NRPs."
Second, the researchers created "dereplication" tools for moving the other direction: taking the chemical structures of known NRPs and other related information and determining what the data signature would look like if a mass spectrometer had blown the compound part.
"Natural products have a long history in therapeutic development and many were discovered before the digital recording of mass spectrometry data. Therefore, we do not have an extensive mass spectrometry database for natural products as we do for proteomics. Our new tools enable dereplication without an experimental database to compare to," said Pieter Dorrestein, assistant professor in the UC San Diego Skaggs School of Pharmacy and Pharmaceutical Sciences and the Departments of Pharmacology, Chemistry and Biochemistry.
By using these two approaches, the researchers have created tools that enable researchers to both characterize the compound they have isolated and check to see if it, or something similar, has been previously described. With dereplication, researchers can leverage known information and are not forced to start from scratch each time a new compound needs to be identified.
"As long as the structure of the therapeutic or a related therapeutic or natural product is in the library, we can accurately dereplicate the molecule. This is the first generation of algorithms that can accomplish this and is a glimpse into the future of modern drug discovery."
Performing de novo sequencing without knowing amino acid masses is completely novel, according to Bandeira. "Until we created them, there were no algorithmic approaches available to do this from mass spectrometry data and it was generally thought to be impossible," said Bandeira, who earned his Ph.D. in computer science from the UC San Diego Jacobs School of Engineering.
The work allows mass spectrometry to go into the natural products field and actually do the identification and characterization of natural products in a high throughput fashion, explained Ng, a bioinformatics PhD student advised by Pavel Pevzner in computer science and Pieter Dorrestein in the Skaggs School of Pharmacy.
The researchers note that currently there is no one place to look for known NRPs, a situation they are trying to change with a new data repository effort.
The UC San Diego web-based tools for sequencing nonribosomal peptides (at not cost to researchers) are available at: bix.ucsd.edu/nrp
"This new study has shown that marine cyanobacteria are incredible sources of new molecules that may have medical value, especially in cancer, infectious diseases and neurological disorders," said Gerwick.
This project was supported by US National Institutes of Health grants 1-P41-RR024851-01, GM086283 and cA10u851, and by the PhRMA foundation.
Journal reference:
Julio Ng et al. Dereplication and De Novo Sequencing of Nonribosomal Peptides. Nature Methods, July 13, 2009
Adapted from materials provided by University of California - San Diego, via EurekAlert!, a service of AAAS.

'Rosetta Stone' Of Bacterial Communication Discovered

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ScienceDaily (July 13, 2009) — The Rosetta Stone of bacterial communication may have been found. Although they have no sensory organs, bacteria can get a good idea about what's going on in their neighborhood and communicate with each other, mainly by secreting and taking in chemicals from their surrounding environment. Even though there are millions of different kinds of bacteria with their own ways of sensing the world around them, Duke University bioengineers believe they have found a principle common to all of them.

The researchers said that a more complete understanding of communication between cells and bacteria is essential to the advancement of the new field of synthetic biology, where populations of genetically altered bacteria are "programmed" to do certain things. Such re-programmed bacterial gene circuits could see a wide variety of applications in medicine, environmental cleanup and biocomputing.
It is already known that a process known as "quorum sensing" underlies communication between bacteria. However, each type of bacteria seems to have its own quorum-sensing abilities, with tremendous variations, the researchers said.
"Quorum sensing is a cell-to-cell communication mechanism that enables bacteria to sense and respond to changes in the density of the bacteria in a given environment," said Anand Pai, graduate student in bioengineering at Duke's Pratt School of Engineering. "It regulates a wide variety of biological functions such as bioluminescence, virulence, nutrient foraging and cellular suicide."
The researchers found that the total volume of bacteria in relation to the volume of their environment is a key to quorum sensing, no matter what kind of microbe is involved.
"If there are only a few cells in an area, nothing will happen," Pai said. "If there are a lot of cells, the secreted chemicals are high in concentration, causing the cells to perform a specific action. We wanted to find out how these cells know when they have reached a quorum."
Pai and scientist Lingchong You, assistant professor of biomedical engineering and a member of Duke's Institute for Genome Sciences & Policy and Center for Systems Biology, have discovered what they believe is a common root among the different forms of quorum sensing. In an article in the July 2009 issue of the journal Molecular Systems Biology, they term this process "sensing potential."
"Sensing potential is essentially the linking of an action to the number of cells and the size of their environment," You said. "For example, a small number of cells would act differently than the same number of cells in a much larger space. No matter what type of cell or their own quorum sensing abilities, the relationship between the size of a cell and the size of its environment is the common thread we see in all quorum sensing systems.
"This analysis provides novel insights into the fundamental design of quorum sensing systems," You said. "Also, the overall framework we defined can serve as a foundation for studying the dynamics and the evolution of quorum sensing, as well as for engineering synthetic gene circuits based on cell-to-cell communications."
Synthetic gene circuits are carefully designed combinations of genes that can be "loaded" into bacteria or other cells to direct their actions in much the same way that a basic computer program directs a computer. Such re-programmed bacteria would exist as a synthetic ecosystem.
"Each population will synthesize a subset of enzymes that are required for the population as a whole to produce desired proteins or chemicals in a coordinated way," You said. "We may even be able to re-engineer bacteria to deliver different types of drugs or selectively kill cancer cells"
For example, You has already gained insights into the relationship between predators and prey by creating a synthetic circuit involving two genetically altered lines of bacteria. The findings from that work helped define the effects of relative changes in populations.
The research was supported by National Institutes of Health, a David and Lucile Packard Fellowship, and a DuPont Young Professor Award.
Adapted from materials provided by Duke University.

DNA Patterns Of Microbes

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ScienceDaily (July 13, 2009) — The genomes or DNA of microbes contain defined DNA patterns called genome signatures. Such signatures may be used to establish relationships and to search for DNA from viruses or other organisms in the microbes' genomes. Foreign DNA in bacteria has often been associated with disease-causing abilities.

In his doctorate, Jon Bohlin studied methods for examining the genome signatures of microbes. Since foreign DNA in the genomes of bacteria often give the bacteria disease-causing abilities, part of his work was aimed at developing fast and simple methods for finding foreign DNA.
The explosive development in technology for sequencing DNA molecules has made enormous amounts of genetic information available for analysis. This has both led to an upheaval in biological research and simultaneously created a great need for fast and effective methods of interpreting the steadily increasing amounts of information.
To solve the challenges that these large amounts of information present, bioinformational research is utilising techniques taken from statistical, mathematical and information technologies. Most of the methods that were used in this project were originally established in the field of theoretical bioinformation. However, because of insufficient information, it was not previously possible to investigate the methods properly.
The increasing number of sequenced genomes that has become available during recent years has made it possible to test the methods' advantages and disadvantages, possibilities and limitations. This has given us more reliable information on how different microbes' DNA composition is influenced by environment and lifestyle.
The methods can also be used to deepen our understanding of the evolutionary development that follows natural selection at the DNA level. Such knowledge is absolutely necessary to understand mechanisms leading to bacteria becoming pathogenic (disease-producing) and resistant to antibiotics.
This doctorate comprises analyses of the genome signatures of microbes, and describes how genome signatures vary in the genomes of both closely-related microbes and among different microbial genomes. One of the central questions is how environment influences the genome signatures and if this influence may be may be linked to different characteristics of the microbes, such as size, DNA composition, lifestyle and niche.
Cand. scient. Jon Bohlin defended his Ph. D. thesis, entitled "Genomic signatures in prokaryotic genomes", at the Norwegian School of Veterinary Science, on June 5, 2009.
Adapted from materials provided by Norwegian School of Veterinary Science.

Key To Maintaining Embryonic Stem Cells In Lab

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ScienceDaily (July 13, 2009) — In a new study that could transform embryonic stem cell (ES cell) research, scientists at UT Southwestern Medical Center have discovered why mouse ES cells can be easily grown in a laboratory while other mammalian ES cells are difficult, if not impossible, to maintain.

If the findings in mice can be applied to other animals, scientists could have an entirely new palette of research tools to work with, said Dr. Steven McKnight, chairman of biochemistry at UT Southwestern and senior author of the study appearing in the July 9 issue of Science Express.
"This might change the way medical research is done. But it's still a big 'if,'" he said.
According to the research, the activation of a gene called TDH in mouse ES cells results in the cells entering a unique metabolic state that is similar to that of rapidly growing bacterial cells. The gene controls the production of the threonine dehydrogenase (TDH) enzyme in mouse ES cells. This enzyme breaks down an amino acid called threonine into two products. One of the two products goes on to control a cellular process called one carbon metabolism; the other provides ES cells with an essential metabolic fuel.
Both of the threonine breakdown products are necessary to keep the ES cells growing and dividing rapidly in a petri dish without differentiating into specific tissues.
The various substances currently used by scientists to keep mouse ES cells alive in the laboratory were found by trying many different combinations until something worked, Dr. McKnight said. But until now, it wasn't known that these culture conditions keyed into keeping the TDH gene actively expressed.
"Scientists added this and that until they got the right 'soup,' one that works in the mouse ES cells to somehow activate the TDH gene," he said, adding that exactly how that gene is regulated is still unknown.
Other mammalian species have a functional version of the TDH gene, suggesting the possibility that the process could also be activated in them.
"You would think that the 'mouse soup' would then work for all species, but it doesn't. Researchers have been trying for 20 years to get the right formula for maintaining ES cells from other species. With few exceptions, however, they still haven't gotten it right," Dr. McKnight said.
The research was funded by a National Institutes of Health Director's Pioneer Award, which Dr. McKnight received in 2004. The program encourages investigators to take on creative, unexplored avenues of research that carry a relatively high potential for failure but that also possess a greater chance for truly groundbreaking discoveries.
"By applying a highly innovative technique to manipulate the TDH gene, McKnight's work could be an important breakthrough with a profound impact on future research," said Dr. Raynard S. Kington, acting director of the NIH. "This research, which was partially funded by our Pioneer Award program, shows the value of supporting exceptionally creative approaches to major challenges in biomedical and behavioral research."
Embryonic stem cells are "blank slate" cells – derived from embryos – that go on to develop into any of the more than 200 types of cells in the adult body.
Because mouse ES cells are easily maintained in the lab, they can be manipulated genetically to produce adult mice in which various genes are either modified or eliminated. So-called "knockout mice" allow scientists to study the genetic aspects of many diseases and conditions, including cancer, Alzheimer's, Parkinson's and paralysis.
In the living mouse, and in other species, ES cells exist for only a short time. In that time, they need to grow rapidly in order to accumulate enough cells to begin the process of differentiating into all the body's cell types. Dr. McKnight hypothesizes that the TDH gene tightly controls this process in the animal, allowing the ES cells to grow, but then it shuts off when it's time to differentiate.
"If we can tweak conditions and determine how to keep the gene turned on in other animals, we might be able to grow and maintain ES cells for study in many species. It's still speculative at this point whether it will work, but if it does, then this may prove to represent a transformational discovery," Dr. McKnight said.
Interestingly, although humans carry a form of the TDH gene, it contains three inactivating mutations. As such, human ES cells do not produce the TDH enzyme.
"In the human embryo, something else is taking the place of this TDH-mediated form of rapid cell growth," Dr. McKnight said. "Human ES cells may exist in a unique metabolic state, but it would not appear to involve threonine breakdown."
Human ES cells grow slowly and are difficult to maintain in the laboratory, which is a huge impediment to this field of study, Dr. McKnight said.
"If scientists could repair the mutated human TDH gene and replace it into human ES cells, could they make those cells grow faster in culture? I don't know whether this will work or not – it's highly speculative. But if so, it would be profound," he said.
Other UT Southwestern researchers involved in the study were lead author Dr. Jian Wang, postdoctoral researcher in biochemistry; Peter Alexander, graduate student in biochemistry; Leeju Wu, senior research scientist in biochemistry; Dr. Robert Hammer, professor of biochemistry; and Dr. Ondine Cleaver, assistant professor of molecular biology.
Adapted from materials provided by UT Southwestern Medical Center.

New Insights Into Formation Of The Centromere, A Key Cellular Structure In Powering And Controlling Chromosome Segregation

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ScienceDaily (July 13, 2009) — Lars Jansen* has described the formation of the centromere, a key cellular structure in powering and controlling chromosome segregation and accurate cell division.

A new Nature Cell Biology paper, published in collaboration with a group at Stanford University School of Medicine, provides insights into the scaffold of proteins that ensures accurate segregation of chromosomes during cell division - a fundamental step to ensure that daughter cells have the same genetic information as their mother, with reduced risk of cancer.
When segregating, chromosomes attach and move along proteins tracks (the mitotic spindle), from the centre of the cell to the poles. The centromere is the area of the chromosome that directs this attachment by controlling the assembly of a scaffold of proteins (called the kinetochore), which tether the chromosome to the spindle, and power its movement along the protein track. The location of the centromere on the chromosome is marked by the presence of a protein, called CENP-A, but how this protein is recognised by the other components of the cell to orchestrate the assembly of the centromere was not understood - until now.
Using a newly developed assay, Lars and his colleagues were able to identify the protein that triggers the assembly of the centromere. It's called CENP-N. According to Mariana Silva, a PhD student in the lab, 'When we depleted CENP-N in cells, the centromere did not assemble correctly and chromosomes segregated abnormally, leading to situations similar to cancer'.
This study shows the applicability of this new assay and open doors to future studies into centromere assembly and structure.
*Lars Jansen moved from California to the Instituto Gulbenkian de Ciência (IGC), in Portugal, last year to head the Epigenetic Mechanisms group.
An EMBO installation grant, of 50,000 euro per year, for a maximum of five years has been awarded.
Adapted from materials provided by Instituto Gulbenkian de Ciencia, via EurekAlert!, a service of AAAS.

Using nuclear magnetic resonance (NMR) methods to determine the structure of the largest membrane-spanning protein to date.

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ScienceDaily (July 12, 2009) — In a landmark technical achievement, investigators in the Vanderbilt Center for Structural Biology have used nuclear magnetic resonance (NMR) methods to determine the structure of the largest membrane-spanning protein to date.

Although NMR methods are routinely used to "take molecular pictures" of small proteins, large proteins – and particularly those that reside within the cell membrane – have been reluctant to smile for the camera.
In the June 26 issue of Science, Charles Sanders, Ph.D., professor of Biochemistry, and colleagues report the NMR structure of the large bacterial protein diacylglycerol kinase (DAGK), a complex of three subunits that each cross the membrane three times (for a total of nine membrane spans).
The group's ability to determine the NMR structure of DAGK suggests that similar methods can now be used to study the structures of other membrane proteins.
"We're taking the methods that we used for diacylglycerol kinase and applying them to high value targets such as G protein-coupled receptors," Sanders said.
G protein-coupled receptors – the largest family of cell signaling proteins – are targets for about half of all pharmaceuticals. Sanders is collaborating with other Vanderbilt investigators to tackle G protein-coupled receptor structure using both NMR and a complementary structural approach, X-ray crystallography.
DAGK may be a therapeutic target for certain types of bacterial infections. It is a virulence factor in the bacteria Streptococcus mutans, which causes tooth decay.
Sanders selected DAGK as a model for studying membrane enzymes when he started his own research lab 17 years ago. DAGK is the smallest known kinase (a protein that adds chemical groups called phosphates onto other molecules), and it is not similar to any other known proteins.
The DAGK structure, Sanders said, "confirmed that this is a really strange kinase." The enzyme has a porch-like structure, with a wide opening for its substrate diacylglycerol and the active site at the top of the porch.
"The active site looks nothing like any other kinase active site – it's a unique architecture," Sanders said.
The researchers also performed exhaustive mutagenesis studies in which they characterized mutations at each amino acid in DAGK and used the data to map the active site of the enzyme onto the structure. They identified two sets of mutations that resulted in non-functional DAGK. One set altered the active site so that it no longer did its job, and the second set caused the protein to fold incorrectly (misfolding).
Sanders said the team was surprised to find that nearly all of the mutations that caused misfolding were in the active site. The expectation, he explained, is that mutations in the active site would cause a loss of function but would not usually affect protein folding, whereas key residues for folding would be located elsewhere in the protein to underpin the scaffold for the active site.
"Our study shows that you can't make that assumption," he said.
Sanders cautions that investigators cannot simply predict the impact of a mutation based on it being located in the active site. The finding has implications for personalized medicine, which aims to use the predicted impact of disease-causing mutations to make therapy decisions.
"The therapeutic strategy for addressing catastrophic misfolding versus simple loss of function may be very different," Sanders said.
Sanders and his team, who got interested in protein folding because of their work with DAGK, are now pursuing structural studies of misfolded membrane proteins that cause diseases including peripheral neuropathy (Charcot-Marie-Tooth Disease), diabetes insipidus and Alzheimer's disease.
"For proteins that misfold because of mutations, we're using NMR tools to understand exactly what the mutations do to the proteins in terms of structure and stability," Sanders said. "We believe that understanding will lead to predictions about how to intervene and avoid misfolding."
Co-authors include Wade Van Horn, Ph.D., Hak-Jun Kim, Ph.D., Charles Ellis, Ph.D., Arina Hadziselimovic, Endah Sulistijo, Ph.D., Murthy Karra, Ph.D., and Changlin Tian, Ph.D., at Vanderbilt and Frank Sönnichsen, Ph.D., at Christian Albrechts University in Kiel, Germany.
Adapted from materials provided by Vanderbilt University Medical Center, via EurekAlert!, a service of AAAS.