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Wayne State Researchers Report a Possible 'Proofreading' Step in DNA Synthesis by Observing Single Molecules of DNA Polymerase
Date:12/3/2009

Research could lead to an understanding of how accurate DNA replication occurs to prevent mutations known to result in cancer and genetic diseases

DETROIT, Dec. 3 /PRNewswire-USNewswire/ -- Wayne State researchers have for the first time observed the movement of a single molecule of DNA polymerase as it incorporates nucleotides one by one. More significantly, they have identified a previously unknown step that they speculate is part of the proofreading process known to prevent the polymerase from producing cancer-causing mutations.

Louis Romano, Ph.D., professor of chemistry, and David Rueda, Ph.D., assistant professor of chemistry in the College of Liberal Arts and Sciences, were published in the Dec. 4, 2009 early edition of the Proceedings of the National Academy of Sciences for their observation. In addition, Thomas Christian, a former WSU chemistry graduate student who is now a postdoctoral associate at Yale University, played a key role in the study.

During DNA replication, DNA polymerase incorporates nucleotides into a DNA chain using the strand of the parental DNA as a template to produce two identical double-stranded product DNA molecules. To maintain proper function of the daughter cells, it is crucial that this process be carried out accurately, and the structure of DNA polymerases have evolved so that they only make a mistake once in ~100,000 nucleotides incorporated. An integral part of this process is the ability of most DNA polymerase to remove incorrectly incorporated nucleotides in a process referred to as proofreading. Rueda and Romano believe that they have identified a new step in the proofreading process that helps maintain the incredible accuracy of DNA replication. This process prevents mutations that can lead to potentially deadly diseases such as cancer.

"DNA polymerase has the extremely important job of building DNA accurately," Romano said. "It is fundamentally important to understand how polymerase incorporates nucleotides, so that we may also understand why they sometimes make mistakes that cause genetic mutations and oftentimes disease."

As part of the movement of the DNA polymerase as it traveled across a DNA template, Romano and Rueda observed the polymerase oscillating between the position where nucleotides are attached, and a position where it possibly "checks" that the correct nucleotide was incorporated.

The discovery was achieved using single-molecule microscopy, a method that Rueda brought to Wayne State that allows researchers to observe the movement of single molecules in real time.

"Scientists have been trying to understand the mechanism by which DNA polymerase acts for more than 60 years," Rueda said. "It is well established that proofreading is a part of this process, but the step we see in our experiments has never before been observed," Rueda said.

The next challenge for Rueda and Romano is to further define this new step in DNA replication and acquire more evidence for its proofreading function. They also hope to observe a polymerase making a mistake during replication - something they have not yet observed because it happens so rarely.

"These studies represent the first step in a long-range program that we hope will greatly improve our understanding of an enzyme vital to maintaining our genetic information and preventing diseases such as cancer," Rueda said.

The full paper can be viewed at http://www.pnas.org/content/early/2009/12/01/0908640106.full.pdf.

Wayne State University is one of the nation's pre-eminent public research universities in an urban setting. Through its multidisciplinary approach to research and education, and its ongoing collaboration with government, industry and other institutions, the university seeks to enhance economic growth and improve the quality of life in the city of Detroit, state of Michigan and throughout the world. For more information on research at Wayne State University, visit http://www.research.wayne.edu.

SOURCE Wayne State University


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SOURCE Wayne State University
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