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Environmental Factor, April 2015

Intramural papers of the month

By Tara Ann Cartwright, John House, Vijay More, and Qing Xu

Sensitive tool to track replication enzymology

In a recent paper published in Nature Structural and Molecular Biology, NIEHS researchers and their collaborators demonstrated a novel high-resolution method for tracking in vivo replication enzymology. Taking advantage of ribonucleotide inclusion in eukaryotic DNA as markers of replication, they devised a new 5’ DNA end-mapping method, called hydrolytic end sequencing (HydEn-seq).

Researchers encoded ribonucleotide excision repair (RER)-deficient and RER-proficient yeast strains for wild type or variant DNA polymerase, with differential ribonucleotide inclusion abilities. They subjected genomic DNA from these strains to alkaline hydrolysis, then prepared libraries of single-stranded DNA fragments for Illumina HiSeq2500 sequencing.

The HydEn-seq technique could be applicable to enzymatic DNA hydrolysis or other lesions in DNA. The authors also project the usefulness of this technique for sensing polymerization changes due to endogenous or exogenous environmental stress. Moreover, with the availability of engineered replicases favoring ribonucleotide inclusion, HydEn-seq could also be made applicable to identifying replication origins, termination zones, and polymerase usage in organisms besides yeast. (VM).

CitationClausen AR, Lujan SA, Burkholder AB, Orebaugh CD, Williams JS, Clausen MF, Malc EP, Mieczkowski PA, Fargo DC, Smith DJ, Kunkel TA. Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation. Nat Struct Mol Biol 22(3):185-191.

A Notch or two in ozone susceptibility

According to NIEHS scientists, mice deficient in either the Notch3 or Notch4 gene exhibited an increased immune response following an ozone-induced airway injury. The work is of vital importance, because ozone, which is one of the major components of smog, is involved in airway inflammation and exacerbation of existing airway diseases, including asthma and chronic obstructive airway disease.

To understand how genetic variation in humans may affect susceptibility to ozone-induced airway inflammation, the research team followed up on prior work that identified, in mice, a region on chromosome 17 associated with ozone-induced pulmonary inflammation. This region included Notch3 and Notch4, two genes known for their roles in development and cell fate decisions.

The researchers found that after ozone injury, the increased immune response displayed by Notch3- or Notch4-deficient mice was characterized by increases in lung permeability, lung neutrophils, and expression of Tnf when compared to similarly exposed wild type mice. In addition, transcriptome analysis revealed important differentially expressed genes, by genotype, related to inflammatory pathways.

This novel work identifies Notch3 and Notch4 genes as susceptibility genes for ozone-induced pulmonary injury, and suggests that Notch receptors, in addition to their well-characterized developmental functions, also function to modulate inflammatory innate immune responses to the environmental toxicant ozone. (JH)

CitationVerhein KC, McCaw Z, Gladwell W, Trivedi S, Bushel PR, Kleeberger SR. 2015. Novel roles for Notch3 and Notch4 receptors in gene expression and susceptibility to ozone-induced lung inflammation in mice. Environ Health Perspect; doi:10.1289/ehp.1408852 [Online 6 February 2015].

Plasma antioxidants are not feasible biomarkers of endotoxin-induced oxidative damage

NIEHS researchers and collaborators report that plasma antioxidants fail to measure the response to oxidative stress induced by endotoxin lipopolysaccharide (LPS). The study provides the latest information on the NIEHS Biomarkers of Oxidative Stress (BOSS) initiative.

The goal of the BOSS initiative is to test if certain blood products could be used as noninvasive indicators of oxidative stress that is involved in various human diseases. Previous studies exposing rats to ozone or to liver-damaging carbon tetrachloride were unable to validate plasma antioxidants as effective markers in those models.

To investigate the change of plasma antioxidants after endotoxin-induced oxidative stress, researchers in this study used the inflammatory LPS to treat Gottingen mini-pigs. Compared to rodents, pigs respond more like humans and serve as their own pretreatment controls. The scientists injected animals with two doses of LPS (2.5 and 5 micrograms/kilogram) and collected blood at four time points (2, 16, 48, and 72 hours). Several plasma antioxidants including ascorbic acid, uric acid, vitamin E, glutathione, and total antioxidant capacity were quantified. Though LPS induced tissue damage, as expected, no significant loss of plasma antioxidants was identified. The data add to the previous findings that plasma antioxidants are not a feasible measurement of oxidative stress. (QX)

CitationKadiiska MB, Peddada S, Herbert RA, Basu S, Hensley K, Jones DP, Hatch GE, Mason RP. 2015. Biomarkers of oxidative stress study VI. Endogenous plasma antioxidants fail as useful biomarkers of endotoxin-induced oxidative stress. Free Radic Biol Med 81:100-106.

Poly beta and FEN1 perform a complementation role in APTX-deficient cells

Researchers from NIEHS have examined an alternative mechanism in which two base excision repair (BER) enzymes — DNA polymerase beta (pol beta) and flap endonuclease 1 (FEN1) — coordinate to remove the 5’-adenylated-deoxyribose phosphate (5’-AMP-dRP) group from BER intermediates in aprataxin (APTX)-deficient cells. APTX removes 5’-AMP-dRP from DNA following abortive ligation attempts by DNA ligase, thereby permitting later repair attempts to protect genomic integrity. APTX mutations cause the autosomal recessive neurodegenerative disease ataxia with oculomotor apraxia type I (AOAI).

The study used whole cell extracts, derived from APTX-null chicken DT40 cells and APTX-deficient AOAI human lymphocytes, to demonstrate that the enzymatic activities of pol beta and FEN1 were strong enough to substitute for APTX activity. The robust activity of pol beta and FEN1 in resolving 5’-AMP-dRP-containing BER intermediates also required coordination with each other. This study also showed that other enzymes with dRP lyase activity, such as DNA pol lambda, pol iota, pol theta, and Ku70, removed 5’-AMP-dRP groups from BER intermediates, but had minimal activity compared with those of pol beta and FEN1.

Taken together, this work highlights the impact of pol beta and FEN1 in the removal of adenylated BER intermediates in the absence of APTX activity. Additional studies are needed to understand how these BER enzymes influence disease phenotypes associated with APTX deficiency. (TAC)

CitationCaglayan M, Horton JK, Prasad R, Wilson SH. Complementation of aprataxin deficiency by base excision repair enzymes. Nucleic Acids Res 43(4):2271-2281.

(Tara Ann Cartwright, Ph.D., is a former postdoctoral fellow in the NIEHS Intracellular Regulation Group. John House, Ph.D., is an Intramural Research Training Award (IRTA) fellow in the NIEHS Genetic Epidemiology Group. Vijay More, Ph.D., is a visiting fellow in the NIEHS Intracellular Regulation Group. Qing Xu is a biologist in the NIEHS Metabolism, Genes, and Environment Group.)

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