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Environmental Factor, October 2012

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Novel technique for determining DNA damage effects on gene transcription

By Sheila Yong

Laura Niedernhofer

Niedernhofer hopes that a better understanding of the genome-level mechanisms in aging will ultimately lead to preventive strategies. (Photo courtesy of Laura Niedernhofer)

A new study funded in part by NIEHS may make it possible to determine exactly how a particular DNA lesion alters transcription and gene expression, more quickly and accurately than previously possible. Published online Aug. 19 by Nature Chemical Biology, the study was conducted by Outstanding New Environmental Scientist (ONES) awardee Laura Niedernhofer, M.D., Ph.D., a team from the University of California, Riverside, and a researcher from the National Institute on Alcohol Abuse and Alcoholism, describing a new competitive transcription and adduct bypass (CTAB) assay.

Niedernhofer, who was on the faculty of the University of Pittsburgh when the paper was submitted, is currently an associate professor in the Department of Metabolism and Aging at the Jupiter, Fla. Campus of the Scripps Research Institute.

“This is a really important line of investigation,” Niedernhofer said of the study. “Most cells in the body are post-mitotic, meaning that they don’t replicate their genome and divide. In these cells, it is of greater concern how an RNA polymerase copes with DNA damage during transcription than a DNA polymerase during replication. We simply don’t know much about the impact of DNA damage on transcription.”

Direct measurement of altered transcripts

When Niedernhofer visited NIEHS in July, she discussed her research on the relationship between DNA damage and aging in a seminar that attracted some of the top researchers at the Institute (see related story). Her research showed that the heterodimeric nuclease ERCC1-XPF promotes efficient DNA damage repair, which is crucial in preventing premature aging. She also discussed her study on the activation of NF-kb in response to DNA damage during accelerated and normal aging, which was published in the July issue of the Journal of Clinical Investigation.

According to Niedernhofer, current techniques for measuring perturbations in the transcription of a lesion-containing DNA template often involve extensive DNA sequencing and colony screening procedures, and do not directly prove that DNA damage alters transcription. The new CTAB assay, on the other hand, enables researchers to quantitatively determine how site-specific DNA lesions affect the efficiency and fidelity of gene transcription. This assay is more efficient since it omits the exhaustive screening steps that most existing protocols require. More importantly, it enables a more accurate identification of mutant transcripts via the use of liquid chromatography and mass spectrometry.

Insights into damage recognition and DNA repair linked to aging

This new technique measures the degree to which a specific DNA adduct inhibits transcription by calculating a relative bypass efficiency (RBE) value. This value represents the ability of an RNA polymerase or cell to polymerize RNA through a damaged DNA template base to  complete gene transcription; the higher the RBE values, the better the cells are at ignoring the damage and carrying on with their job of making mRNA. In this study, the researchers examined how DNA adducts affect transcription. These adducts arise in the mammalian genome as a result oxidative insults, exposure to environmental contaminants, and normal physiological processes. If left unrepaired, they contribute to various cancers, health complications, and aging.

The key findings of the study were that several of these oxidative DNA lesions, which are abundant in our bodies, are strong inhibitors of transcription in mammalian cells, including N2-CEdG, S-cdA, and S-cdG. In addition, several lesions caused the polymerases to make mistakes and introduce mutations into the transcript, including S-cdA and S-cdG. These findings provide novel evidence that DNA damage can have profound effects even on non-replication cells, potentially leading to production of fewer or erroneous proteins.

Niedernhofer and her colleagues conclude that future work should be aimed at expanding the repertoire of lesions studied in the CTAB assay to discover the effects of environmental genotoxins and chemotherapeutic agents on transcription and non-dividing cells.

Citation: You C, Dai X, Yuan B, Wang J, Wang J, Brooks PJ, Niedernhofer LJ, Wang Y. 2012. A quantitative assay for assessing the effects of DNA lesions on transcription. Nat Chem Biol: doi: 10.1038/nchembio.1046 [Online 19 August 2012].

(Sheila Yong, Ph.D., is a visiting fellow in the NIEHS Laboratory of Signal Transduction.)

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