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Intramural papers of the month

By Jeffrey Stumpf and Mamta Behl
November 2010

3D structure of dust mite allergens

NIEHS researchers determined the first three-dimensional structure of Der p 5, an allergenic protein produced by a common house dust mite. Immune reactions to Der p 5 and other group 5 allergens are prevalent among allergic patients worldwide. This study revealed that Der p 5 has structural properties that may be important in eliciting allergenic responses.

Der p 5 was shown to be predominantly a monomer, but was also present as a dimer, hexamer, and dodecamer at higher concentrations. The structure illuminated a large hydrophobic pocket at the dimer interface that the authors suggest may be important for binding hydrophobic compounds. The immune system is hypersensitive to hydrophobic molecules from bacteria, which normally stimulate an inflammatory response needed to deter infections. The erroneous stimulation of the immune system by allergens and their hydrophobic "cargo" is believed to skew the immune response toward allergy and away from normal tolerance.

Allergic disease is a major environmental problem that affects one-fifth of the world population. In particular, extrinsic asthma is strongly associated with sensitivity to house dust mites. Illumination of the structure of this allergen furthers the understanding of its role in the origins of allergic disease and may suggest future routes for therapy.

Citation: Mueller GA, Gosavi RA, Krahn JM, Edwards LL, Cuneo MJ, Glesner J, et al. ( Exit NIEHS 2010. Der p 5 crystal structure provides insight into the group 5 dust mite allergens. J Biol Chem 285(33):25394-25401.

DNA double-strand breaks may play a role in the mutagenesis of antibody diversification

Researchers at NIEHS demonstrated a possible role of DNA double-strand breaks (DSBs) in the localized increased mutagenesis necessary for producing different antibodies during the adaptive immune response. This process, called somatic hypermutation, is promoted by activation-induced deaminase (AID), which initiates the mutagenesis process by converting cytosine to uracil in the immunoglobulin genes that encode the regions of antibodies that bind to antigens.

The authors investigated a potential role of DSBs in somatic hypermutation by making use of an earlier report by the Resnick group at NIEHS to engineer yeast strains with two important features: 1) an inducible DSB site positioned downstream of a mutation reporter gene, and 2) inducible expression of recombinant human AID. The authors found that large increases in reporter gene mutagenesis depended on the concurrent induction of a DSB, the expression of AID, and the presence of DNA polymerase delta. These requirements along with the mutation profile in the reporter gene suggested that the mutations were formed by the ability of the DNA polymerase delta to bypass AID-induced uracil bases. Overall, the results are consistent with a model involving single-strand DNA resection at the site of the DSB, and then DNA polymerase delta re-synthesis to fill-in the gap, restoring the double-stranded DNA. Mutations are introduced during the re-synthesis step.

DSBs are prevalent at the immunoglobulin locus and may localize mutagenesis by triggering resection and the formation of regions of single-strand DNA that are substrates for mutation prone re-synthesis of double-stranded DNA. This model may explain how a dangerously mutagenic and carcinogenic process has been modified for a role in maintaining proper immunity.

Citation: Poltoratsky V, Heacock M, Kissling GE, Prasad R, Wilson SH. ( Exit NIEHS 2010. Mutagenesis dependent upon the combination of activation-induced deaminase expression and a double-strand break. Mol Immunol September 7 doi:10.1016/j.molimm.2010.08.013 [Epub ahead of print]

Genome instability due to ribonucleotide incorporation into DNA

DNA is more stable for storing genetic information than is RNA, because the ribose sugar in RNA is intrinsically more prone to strand cleavage that could led to mutations. Although most DNA polymerases efficiently prevent ribonucleotides from being incorporated into DNA, this exclusion is not absolute. This finding implies that some ribonucleotides will be incorporated into DNA in vivo, and that they need to be removed to maintain the chemical identity of organisms like humans, whose genomes are comprised of DNA.

When researchers at the NIEHS and UmeÅ University in Sweden recently tested these ideas, the results established three important facts about DNA replication:

  • Ribonucleotides are indeed incorporated during replication in vivo
  • The ribonucleotides are normally removed by RNase H2-dependent repair
  • Defective repair causes cellular stress and genome instability.

These findings have potentially important implications for human health. Theoretically, the genome instability resulting from loss of ribonucleotide repair could be relevant to humans with inherited defects in RNase H2. These patients suffer from a severe autoimmune disease, Acardi Goutiéres Syndrome. An RNase H2 defect is also one possible source of genome instability associated with cancer.

Experiments are underway to examine these issues, and to discover the enzymatic mechanisms for the unusual specificity of mutations seen in RNase H2-defective cells. The authors also speculate that the transient presence of ribonucleotides in DNA could have beneficial consequences, for example, in signaling for certain important cellular processes.

Citation: Nick McElhinny SA, Kumar D, Clark AB, Watt DL, Watts BE, Lundström EB, et al. ( Exit NIEHS 2010. Genome instability due to ribonucleotide incorporation into DNA. Nat Chem Biol 6(10):774-781.

Effect of alteration in DNA methylation on lymphomas

Recent data suggests that BCL6 oncogene expression is maintained during lymphomagenesis, partly through DNA methylation that prevents CTCF-mediated silencing; CTCF is an enhancer-blocking transcription factor. The research represents a collaborative effort from scientists at NIEHS, Emory University School of Medicine, and SRA International, Inc. It is the first published work to demonstrate the role that DNA methylation plays in the activation of oncogenes.

Abnormal DNA methylation commonly occurs in cancer cells. Although the role of DNA methylation in gene inactivation to promote formation of tumors is well documented, its role in gene activation, particularly of oncogenes had not been clearly demonstrated.

In lymphoma cells, the researchers demonstrated hypermethylation at a specific site on the BCL6 locus that expressed high amounts of BCL6 messenger RNA (mRNA). Consequently, inhibition of DNA methyltransferases decreased BCL6 mRNA abundance, suggesting a role for methylation in the regulation of BCL6 transcription in these cells. In neoplastic plasma cells that do not express BCL6, there was an up-regulation of BCL6 transcription when enhancer-blocking transcription factor CTCF was depleted by short hairpin RNA.

These findings suggest that the fundamental outcome of DNA methyltransferase activity is to alter chemical properties and information content of the DNA subsequently regulating transcription and chromosomal structure.

Citation: Lai AY, Fatemi M, Dhasarathy A, Malone C, Sobol SE, Geigerman C, et al. ( Exit NIEHS 2010. DNA methylation prevents CTCF-mediated silencing of the oncogene BCL6 in B cell lymphomas. J Exp Med 207(9):1939-1950.

(Jeffrey Stumpf, Ph.D., is a postdoctoral fellow in the NIEHS Laboratory of Molecular Genetics Mitochondrial DNA Replication Group. Mamta Behl, Ph.D., is a research fellow in the NIEHS National Toxicology Program.)

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