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Intramural Papers of the Month

By Laura Hall and Omari Bandele
January 2010

Initiation of Repair of Random DNA Double Strand Breaks Requires RAD50

DNA double-strand breaks (DSBs) are important sources of genome stability and disease. Researchers from NIEHS and Indiana University-Purdue University Indianapolis found a way to track early events in repairing DSBs caused by gamma irradiation in yeast using pulsed field gel electrophoresis (PFGE).

They showed that the initial step in repair of random DSBs involved resection — the removal of one of the strands at a DSB end. This step depends on the MRX complex composed of the Mre11, Rad50, and Xrs2 proteins. Yeast lacking Rad50 or Mre11 were slow in generating resected ends — a key step in DSB repair in most organisms.

While much is known about DSB mechanisms based on model systems employing a unique DSB, little is known about the random breaks created by environmental agents, especially gamma radiation commonly used in cancer radiation treatment. The authors incorporated circular chromosomes into yeast cells, then irradiated them to cause DSBs that changed the chromosomes to a linear form that that could be detected with PFGE.

Surprisingly, the subsequently resected linear chromosomes with single-stranded DNA tails were less mobile in the gel than those without a tail. This mobility shift provided a unique opportunity to examine repair by tracking the appearance of the tails and disappearance of the linearized chromosome as repair was completed.

Citation: Westmoreland J, Ma W, Yan Y, Van Hulle K, Malkova A, Resnick MA. ([Author]&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract)Exit NIEHS 2009. RAD50 is required for efficient initiation of resection and recombinational repair at random, gamma-induced double-strand break ends. PLoS Genet 5(9):e10000656.

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New Pathway for Viral Regulation of the Cell Cycle Through TGF-β

NIEHS researchers have shown that the cytokine, transforming growth factor beta (TGF-β), enhanced respiratory syncytial virus (RSV) replication by inducing cell cycle arrest in human lung epithelial cells. Furthermore, RSV infection induced the expression of TGF-β enhancing its own replication.

Blocking TGF-β with TGF-β antibody or inhibitor, or using a TGF-β receptor signaling inhibitor, prevented the RSV-induced cell cycle arrest. This effect, resulting from signaling cytokine or receptor blockage, suggests a TGF-β autocrine pathway.

RSV, a single stranded RNA virus, is a common cause of severe respiratory infections in children and is associated with the development and exacerbations of asthma. Previous genetic studies have shown an association between asthma phenotype and TGF-β, a cytokine or cell signaling protein involved in regulation of the cell cycle.

TGF-β is also involved in fibrosis, inhibiting inflammation and regulating immune function. Aberrant expression of TGF-β in the lung leads to fibrosis, airway remodeling, and mucus hypersecretion like that found in asthma. The NIEHS study shows a connection between RSV and TGF-β, which suggests a mechanism to explain how RSV infections can lead to the development and exacerbations of an asthma phenotype.

Citation: Gibbs JD, Ornoff DM, Igo HA, Zeng JY, Imani F. ([Author]&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract)Exit NIEHS 2009. Cell cycle arrest by transforming growth factor beta1 enhances replication of respiratory syncytial virus in lung epithelial cells. J Virol 83(23)12424-12431.

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Methoxyacetic Acid Disrupts Endogenous Estrogen Receptor Signaling

A collaborative study by researchers from NIEHS and the German Cancer Research Center in Heidelberg, Germany demonstrates that methoxyacetic acid (MAA), a short-chain fatty acid, exerts antiestrogenic effects by reducing the levels of endogenous estrogen receptor-alpha (ERα). The authors also show for the first time that MAA reduces estrogen-induced expression of ERα target genes.

MAA is the primary metabolite of ethylene glycol monomethyl ether (EGME), an industrial solvent. Exposure to EGME is associated with reproductive toxicity in humans and animals, which is attributed to MAA. Using human cells and mice as models, Korach and colleagues sought to gain insight into the molecular mechanisms of MAA activity on the estrogen-signaling pathway. They report that the MAA-induced antiestrogenic effects observed in the study are similar to those seen with other members of the short-chain fatty acid family, which suggests a common mechanism of action.

The effects of MAA on endogenous ER are consistent with the reproductive abnormalities reported following EGME exposure in humans and animals. These results suggest that MAA-induced attenuation of endogenous ERα signaling may likely contribute to these toxicities.

Citation: Henley DV, Mueller S, Korach KS. 2009. The short-chain fatty acid methoxyacetic acid disrupts endogenous estrogen receptor-α-mediated signaling. Environ Health Perspect 106(11): 1702-1706.

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Structural Studies Reveal Specificity of mRNA Regulatory Proteins

Researchers from NIEHS and the University of Wisconsin recently provided the first structural explanation for the unique specificity of a PUMILIO/fem-3 binding factor (PUF) protein for distinct RNA targets. This study suggests that PUF protein-induced base flipping may be a common mechanism utilized to distinguish specific RNA targets.

Hall and colleagues utilized the RNA-binding domain of a Caenorhabditis elegans PUF protein, FBF-2, to capture protein-RNA complexes. The FBF protein is a founding member of the PUF family of mRNA regulatory proteins. It regulates multiple mRNAs that are critical for stem cell maintenance and germline development.

The authors used six different RNA sequences, including four natural mRNAs to probe these interactions. Through structural and biochemical experiments, the group determined that the positions of flipped and specifically recognized bases within target sequences contribute to FBF-2 sequence specificity.

Together with other PUF protein structural studies, this work provides a model of PUF protein specificity for RNA and its evolution.

Citation: Wang Y, Opperman L, Wickens M, Hall TMT. ( NIEHS 2009. Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein. Proc Natl Acad Sci U. S. A. 106(48):20186-20191.

(Laura Hall is a biologist in the NIEHS Laboratory of Pharmacology currently on detail as a writer for the Environmental Factor. Omari J. Bandele, Ph.D. is a postdoctoral fellow in the NIEHS Laboratory of Molecular Genetics Environmental Genomics Group.)

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