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DNA Repair

Armed with advanced microscopy methods, scientists are watching real-time cellular processes with new detail. These observations are providing important insights into the causes and progression of cancer.

Researchers at the University of California, Davis, recently watched in real time as cellular machinery prepped single strands of DNA for repair. Stephen Kowalczykowski, who led the study, explains that repairing a break in the DNA double helix requires a single strand to find its matching sequence on the opposite strand by looking for where the single strand is coated with the protein RecA.

The researchers wanted to better understand how mediator proteins facilitate this important RecA-coating process because it might reveal more about the processes role in the formation of some cancers. In humans, mutations in the gene coding for the mediator protein BRCA2 are strongly associated with breast cancer.

In the new study, the researchers used single molecule microscopy technology they’ve been developing for the past decade to observe individual strands of bacterial DNA being coated with RecA. They also used optical trapping and atomic force microscopy to analyze the repair process. Their imaging equipment included a Nikon total internal fluoresce microscope, a 488nm Picarro laser, and an Andor CCD camera.

The video shows the DNA repair process, which begins with two molecules of RecA attaching to the DNA. Single molecules of RecA can then be added at either end, similar to adding beads on a string. Videography by Jason Bell and Steve Kowalczykowski.

One of the findings revealed by the imaging study was that when the mediator proteins were absent, the RecA coating process was relatively slow. If the process is too slow, it could lead to improper repair of DNA breaks.

Watching cancer spread

For some time, researchers have used intravital microscopy and surgically implanted windows to watch various cellular processes in living animals such as mice. However, these windows are designed for short-term use and thus aren’t ideal to study slow processes such as cancer spreading to other organs. Researchers from the Netherlands have developed an imaging window that can be used to image abdominal organs for up to a month. As detailed in a new paper, the imaging window allows the observation of biological processes in the spleen, kidney, small intestine, pancreas, and liver.

A small, surgically implanted glass window lets researchers watch the real-time development of cancer in the liver, spleen, kidney and small intestine in mice. Courtesy of Science Translational Medicine/AAAS.

The investigators used the new imaging window to study cancer cell migration during the different phases of liver colonization in mice for 14 days using a Leica two-photon microscope with a Coherent Chameleon Ti:Sapphire laser. They found that single tumor cells multiplied into pre-micrometastases and then condensed into micrometastases where cell migration was strongly diminished but proliferation continued. The researchers say that the mobility of tumor cells in the pre-micrometastases might make this a good stage of liver metastasis formation to target with therapeutics.

Written by Nancy Lamontagne, Contributing Editor, Novus Light Technologies Today

Labels: microscopy,biophotonics,biotech,imaging

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