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August 2011

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New study explores the unexpected interface of RNA and DNA

By Robin Arnette
August 2011

Thomas Kunkel, Ph.D.

Kunkel is a principal investigator in the NIEHS Laboratory of Molecular Genetics and chief of the Laboratory of Structural Biology. He said that finding ribonucleotides in DNA as a cause by which mutations arise in the genome was a novel finding that took everyone by surprise. (Photo courtesy of Steve McCaw)

Jessica Williams, Ph.D.

“Although the idea is pure speculation at the moment, it's conceivable that RNase H2 defects are also relevant to autoimmunity in general,” Williams added. (Photo courtesy of Steve McCaw)

biologist Alan Clark

Alan Clark is a co-author on the article published in Science magazine. (Photo courtesy of Steve McCaw)

A group of NIEHS researchers, led by Thomas Kunkel, Ph.D., recently made a surprising find. They demonstrated that the enzymes in yeast that replicate or copy DNA in eukaryotes incorporate lots of ribonucleotides, which are normally part of RNA, not DNA.

Kunkel's results appeared online( Exit NIEHS in the March 1, 2010 issue of the Proceedings of the National Academy of Sciences (PNAS), but the article raised more questions, which Kunkel pursued in his latest work( Exit NIEHS, published in the June 24 issue of Science.

Kunkel was intrigued by the implications of having ribonucleotides involved in DNA replication stability. For instance, since ribonucleotides are sensitive to strand breakage, did the presence of ribonucleotides in DNA lead to mutagenesis or genome instability? The answers had potential implications for understanding the origins of human disease, so Kunkel was compelled to keep digging for clues.

His latest study showed that removing the ability of yeast cells to get rid of ribonucleotides in DNA creates an uncommon mutation in the DNA, but the mutation could be abolished if the activity of topoisomerase 1 (Top1), an enzyme that maintains the integrity of DNA, was lost. This novel breakthrough not only has implications for cancer research; it also fosters a new way of thinking about the possible causes of a rare autoimmune disease called Aicardi Goutiéres syndrome( Exit NIEHS and perhaps other autoimmune disorders.

RNase H2 and Top1

Kunkel's research fellow, Jessica Williams, Ph.D., and biologist Alan Clark, constructed two strains in budding yeast. In one strain, they deleted the gene that encodes the catalytic subunit of RNase H2, a key enzyme that removes ribonucleotides from DNA. In the other, they deleted the genes for RNase H2and Top1. Using a reporter system that monitored spontaneous mutations that occur in DNA, they found that the yeast lacking RNase H2 exhibited short, unusual 2-5 base pair deletions in repetitive sequences. However, the strain missing both RNase H2 and Top1 didn't display these mutations. The data suggested that the deletions were due to the activity of Top1.

“These results are meaningful because chemicals that inhibit Top1 activity are used as anti-cancer agents, and mutations in the RNase H2 enzyme have been identified in patients with Aicardi Goutiéres syndrome,” Williams said.

Peeling the research onion

Just like Kunkel's PNAS article and subsequent papers, these findings pose more questions. He said a few obvious inquiries come to mind. Are ribonucleotides in DNA involved in tumor formation? Do ribonucleotides in DNA have a signaling function? Are there additional DNA repair pathways outside of RNase H2 that process ribonucleotides in DNA? His group is currently examining several of these ideas.

“The cell could have many things going on that depend on ribonucleotides in DNA that no one has explored,” Kunkel noted.

Two worlds of thought come together

Although Kunkel's studies on ribonucleotides in DNA laid the groundwork for this article, his collaborators, Sue Jinks-Robertson, Ph.D., of Duke University, and Yves Pommier, M.D., Ph.D.,( Exit NIEHS of NIH's National Cancer Institute, also uncovered key data. Jinks-Robertson's previously published work generated the same rare 2-5 base pair deletion in yeast, but the culprit wasn't ribonucleotides in DNA; rather it correlated with a high level of gene transcription known as transcription-associated mutagenesis. Since these deletions depended on Top1 activity, her group investigated a likely connection between ribonucleotides and Top1.

As a specialist in topoisomerases, Pommier provided direct biochemical evidence that Top1 cleaves at the site of a ribonucleotide causing a break in the DNA, which can lead to the deletion mutations observed in the yeast strains.

Kunkel said the thing that excited him the most about the Science paper was the linkage between DNA replication and transcription. Experts in both fields now need to pay attention to the scientific connections between the two. He summed up his feelings by saying, “[This work] is at this interface between two very big efforts in research by people who don't normally pay a lot of attention to each other. To me, discoveries are made at these kinds of interfaces.”

Citation: Kim N, Huang SY, Williams JS, Li YC, Clark AB, Cho JE, Kunkel TA, Pommier Y, Jinks-Robertson S.( Exit NIEHS 2011. Mutagenic processing of ribonucleotides in DNA by yeast topoisomerase 1. Science 332(6037):1561-1564.

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