First Complete Pictures of Cells' DNA-Copying Machinery

The first-ever images of the protein complex that unwinds, splits, and copies double-stranded DNA reveal something rather different from the standard textbook view. The electron microscope images, created by scientists at the U.S. Department of Energy's Brookhaven National Laboratory with partners from Stony Brook University and Rockefeller University, offer new insight into how this molecular machinery functions, including new possibilities about its role in DNA "quality control" and cell differentiation. The images and implications are described in a paper published online by the journal Nature Structural & Molecular Biology, November 2, 2015.

"This work is a continuation of our long-standing research using electron microscopy to understand the mechanism of DNA replication, an essential function for every living cell," said Huilin Li, a biologist with a joint appointment at Brookhaven Lab and Stony Brook University.

"These new images show the fully assembled and fully activated 'helicase' protein complex—which encircles and separates the two strands of the DNA double helix as it passes through a central pore in the structure—and how the helicase coordinates with the two 'polymerase' enzymes that duplicate each strand to copy the genome."

Three-dimensional structure of the active DNA helicase bound to the front-end DNA polymerase (Pol epsilon). The DNA polymerase epsilon (green) sits on top rather than the bottom of the helicase.

Studying this molecular machinery, known collectively as a "replisome," and the details of its DNA-copying process can help scientists understand what happens when DNA is miscopied—a major source of mutation that can lead to cancer—or learn more about how a single cell can eventually develop into the many cell types that make up a multicellular organism. But no one has produced a real structure of a replisome at any resolution for any organism until now.

"All the textbook drawings and descriptions of how a replisome should look and work are based on biochemical and genetic studies," Li said, likening the situation to the famous parable of the three blind men trying to describe an elephant, each looking at only one part. Those textbook drawings show the helicase moving along the DNA, separating the two strands of the double helix, with two polymerases located at the back where the DNA strand is split. In this configuration, the polymerases would add nucleotides (molecules containing the complementary A, T, G, and C bases of the genetic code) to the side-by-side split ends as they move out of the helicase to form two new complete double helix DNA strands.

Collaborating scientists and study coauthors Zuanning Yuan, a graduate student at Stony Brook University (standing), Huilin Li of Stony Brook and Brookhaven Lab (seated, back), and Jingchuan Sun of Brookhaven Lab (seated, front) examining protein structures.

To test these assumptions, Li's group turned to the technique they had previously used to study individual components of the helicase, electron microscopy (EM). Jingchuan Sun, an EM expert in Li's lab, was essential to the success of the work. He studied samples of replisomes from baker's yeast cells—a model for all nucleus-containing cells—prepared and provided by Roxana Georgescu in Michael O'Donnell's research group at Rockefeller University. O'Donnell's group had previously published biochemical results related to this work.

"DNA replication is one of the most fundamental processes of life, so it is every biochemist's dream to see what a replisome looks like," Sun said. "Our lab has expertise and a decade of experience using electron microscopy to study DNA replication, which has prepared us well to tackle the highly mobile therefore very challenging replisome structure. Working together with the O'Donnell lab, which has done beautiful functional studies on the yeast replisome, our two groups brought perfectly complementary expertise to this project," he said.

The team's first-ever images of an intact replisome revealed that only one of the polymerases is located at the back of the helicase. The other is on the front side of the helicase, where the helicase first encounters the double-stranded helix. This means that while one of the two split DNA strands is acted on by the polymerase at the back end, the other has to thread itself back through or around the helicase to reach the front-side polymerase before having its new complementary strand assembled.

The scientists were so surprised by this finding that they asked another group at Rockefeller, led by Brian Chait, to perform additional structural studies using mass spectrometry. Yi Shi, a postdoctoral fellow in Chait's group performed this work, which confirmed the electron-microscopy-based conclusion about the unexpected architecture of the replisome.

The counterintuitive position of one polymerase at the front of the helicase suggests that it may have an unforeseen function. The authors suggest several possibilities including keeping the two "daughter" strands separate to help organize them during replication and cell division. It might also be possible that, as the single strand moves over other portions of the structure, some "surveillance" protein components check for lesions or mistakes in the nucleotide sequence before it gets copied—a sort of molecular quality control.

This architecture could also potentially play an important role in developmental biology by providing a pathway for treating the two daughter strands differently. Many modifications to DNA, including how it is packaged with other proteins, control which of the many genes in the sequence are eventually expressed in cells. An asymmetric replisome may result in asymmetric treatment of the two daughter strands during cell division—an essential step for making different tissues within a multicellular organism.

As the paper concludes, "Clearly, further studies will be required to understand the functional implications of the unexpected replisome architecture reported here."

This research was funded by the National Institutes of Health (GM103314, GM109824, GM74985, AG29979, and GM38839) and by the Howard Hughes Medical Institute.

Brookhaven National Laboratory

‘Make-or-break’ protein holds key to cancer spread

A potential cancer cell extruding from an epithelium. Courtesy of Selwin Wu, Yap Lab, UQ's Institute for Molecular Bioscience

University of Queensland researchers have discovered a protein in cells that could block the escape route of potentially cancerous cells and stop them spreading to other parts of the body.

A team of biologists, physicists and mathematicians led by Professor Alpha Yap from UQ’s Institute for Molecular Bioscience made the discovery using microscopic imaging and statistical techniques.

“The finding could lead to new targeted treatments for cancer and other diseases,” Professor Yap said.

The researchers revealed and analysed molecular processes that cause potentially cancer-causing cells to escape from epithelial tissues, the layers of cells that cover and protect organs, including skin.

Professor Yap said the team had made important new insights into cancer biology, pinpointing the pathway these cells take to exit the epithelial tissue and investigating how the protein N-WASP can block their escape route.

“Abnormal or dying cells pose a risk to the health of the protective barrier that cells form around our organs,” he said.

“The normal cells that surround these dangerous cells use the complex process of cellular extrusion to push them out of the tissue,” Professor Yap said.

“However, when cancer cells are pushed out, it gives them the opportunity to grow or invade surrounding healthy tissue, which can cause the cancer to spread to other parts of the body and make it harder to control and treat.

“So while our normal cells think they’re doing us a favour by pushing out the bad cells, they’re actually helping the cancer cells to spread,” he said.

Professor Yap said his team had found a way to potentially block the escape routes by inhibiting the protein N-WASP, which regulates the internal skeleton of the cells.

“The pathway that makes or breaks these cells from escaping is regulated by N-WASP,” he said.

“We have found that if we can inhibit N-WASP from functioning, then we can stop these potentially cancerous cells from spreading.”

The study was conducted by researchers from UQ’s Institute for Molecular Bioscience in collaboration with Dr Zoltan Neufeld from UQ’s School of Mathematics and Physics.

The research has been published in the scientific journal Nature Cell Biology and was supported by the National Health and Medical Research Council of Australia, the Australian Research Council, the Kids Cancer Project of The Oncology Children’s Foundation, and The University of Queensland.

Confocal and optical microscopy was performed at the IMB’s ACRF Cancer Biology Imaging Facility, established with the generous support of the Australian Cancer Research Foundation.

The Institute for Molecular Bioscience (IMB) is a research institute of The University of Queensland that aims to improve quality of life by advancing medical genomics, drug discovery and biotechnology.

The UQ School of Mathematics and Physics has an international reputation for cutting-edge research and innovative teaching in mathematics, physics and statistics.

Source link: http://www.uq.edu.au/news/node/112860

Nanopore opens new cellular doorway for drug transport

A living cell is built with barriers to keep things out – and researchers are constantly trying to find ways to smuggle molecules in.‬ ‪Professor Giovanni Maglia (Biochemistry, Molecular and Structural Biology, KU Leuven) and his team have engineered a biological nanopore that acts as a selective revolving door through a cell's lipid membrane. The nanopore could potentially be used in gene therapy and targeted drug delivery.

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All living cells are enclosed by a lipid membrane that separates the interior of the cell from the outside environment. The influx of molecules through the cell membrane is tightly regulated by membrane proteins that act as specific doorways for the trafficking of ions and nutrients. Membrane proteins can also be used by cells as weapons. Such proteins attack a cell by making holes – nanopores – in 'enemy' cell membranes. Ions and molecules leak from the holes, eventually causing cell death.‬‬

‪Researchers are now trying to use nanopores to smuggle DNA or proteins across membranes. Once inside a cell, the DNA molecule could re-programme the cell for a particular action. Professor Maglia explains: "‪We are now able to engineer biological nanopores, but the difficult part is to precisely control the passage of molecules through the nanopores' doorways. We do not want the nanopore to let everything in. Rather, we want to limit entry to specific genetic information in specific cells." ‬‬‬‬‬‬‬‬‬‬‬‬‬‬

‪Professor Maglia and his team succeeded in engineering a nanopore that works like a revolving door for DNA molecules. "We have introduced a selective DNA revolving door atop of the nanopore. Specific DNA keys in solution hybridise to the DNA door and are transported across the nanopore. A second DNA key on the other side of the nanopore then releases the desired genetic information. A new cycle can then begin with another piece of DNA – as long as it has the correct key. In this way, the nanopore acts simultaneously as a filter and a conveyor belt." ‬‬‬‬‬‬‬

"In other words, we have engineered a selective transport system that can be used in the future to deliver medication into the cell. This could be of particular use in gene therapy, which involves introducing genetic material into degenerated cells in order to disable or re-programme them. It could also be used in targeted drug delivery, which involves administering medication directly into the cell. The possibilities are promising."‬‬

 

The researchers' findings were published in a recent edition of Nature Communications.

Source: http://www.kuleuven.be/english/news/nanopore-opens-new-cellular-doorway-for-drug-transport

Great potential for faster diagnoses with new method

The more accurately we can diagnose a disease, the greater the chance that the patient will survive. That is why many researchers are working to improve the quality of the diagnostic process. Researchers at the Nano-Science Center, University of Copenhagen have discovered a method that will make the process faster, cheaper and more accurate. This is possible, because they are combining advanced tools used in physics for research in biology at nanoscale, two scientific disciplines usually very distant from each other.

Many diseases can be diagnosed using so-called biomarkers. There are substances, for example, that can be measured in a blood sample, which shows that the patient is suffering from the disease in question. These biomarkers are often proteins that are found in very small quantities in the blood, making it difficult to detect them. By measuring them, the diagnosing is more precise and many diseases can be detected very early, before the patient develops severe symptoms.

- We have developed a method in which we optimise the analysis of the proteins. A central point of this method is the use of nanowires to hold the proteins while we analyse them. It is unique, explains Katrine R. Rostgaard, a PhD student at the Nano-Science Center, Department of Chemistry, University of Copenhagen.

Researchers normally use small plates to hold the proteins when they need to be analysed, but by using nanowires, which are cylindrical structures having a diameter of about 1/1000th of a human hair, they add a third dimension to the sample. The nanowires stand up like a little forest, creating a much greater surface area to hold the proteins because they can sit on all sides of the nanowire.

- With greater area, we can hold more proteins at once. This makes it possible to measure for multiple biomarkers simultaneously and it increases the signal, thereby providing a better quality of diagnosis, says Katrine R. Rostgaard about the method, which has just been published in the journal Nanoscale.

Profitable method for diagnosingThe research is done at the nanoscale on small size samples. The forests of nanowires are used to capture the proteins they want to study directly. When examining the proteins, you can reuse the nanowires by performing a multiple tests on the same protein. This simplifies the workflow in the laboratory tremendously in comparison to the conventional method, where researchers have to start over with a new plate to hold the proteins every time they perform a new analysis. In this way, the method helps to make the diagnostic process more environmentally friendly and economically viable for use in, for example, industry.

- We know that several major biotech companies will be interested in our new method and find potential applications, though it requires improvements before it is ready for use in the industry, explains Karen Martinez, research group leader of the Nanobio group at the Nano-Science Center, Department of Chemistry, University of Copenhagen. 

The work is part of two larger projects, ANaCell and UNIK Center for Synthetic Biology, which is financed by The Danish Council for Strategic Research and the Ministry of Science, Technology and Innovation. 

Source: http://news.ku.dk/all_news/2013/2013.9/faster-diagnoses-with-new-method/