A functional genomics approach to identify and characterize oxidation resistance genes.

In order to develop a more complete understanding of the genes required for resistance to oxidative DNA damage, we devised methods to identify genes that can prevent or repair oxidative DNA damage. These methods use the oxidative mutator phenotype of a repair deficient E. coli strain to measure the antimutator effect resulting from the expression of human cDNAs. The method can be adapted to characterize the function, and to determine the active site domains, of putative antimutator genes. Since bacteria do not contain subcellular compartments, genes that function in mitochondria, the cytoplasm, or the nucleus can be identified. Methods to determine the localization of genes in their normal host organism are also described.

The OXR domain defines a conserved family of eukaryotic oxidation resistance proteins.

BACKGROUND: The NCOA7 gene product is an estrogen receptor associated protein that is highly similar to the human OXR1 gene product, which functions in oxidation resistance. OXR genes are conserved among all sequenced eukaryotes from yeast to humans. In this study we examine if NCOA7 has an oxidation resistance function similar to that demonstrated for OXR1. We also examine NCOA7 expression in response to oxidative stress and its subcellular localization in human cells, comparing these properties with those of OXR1. RESULTS: We find that NCOA7, like OXR1 can suppress the oxidative mutator phenotype when expressed in an E. coli strain that exhibits an oxidation specific mutator phenotype. Moreover, NCOA7's oxidation resistance function requires expression of only its carboxyl-terminal domain and is similar in this regard to OXR1. We find that, in human cells, NCOA7 is constitutively expressed and is not induced by oxidative stress and appears to localize to the nucleus following estradiol stimulation. These properties of NCOA7 are in striking contrast to those of OXR1, which is induced by oxidative stress, localizes to mitochondria, and appears to be excluded, or largely absent from nuclei. CONCLUSION: NCOA7 most likely arose from duplication. Like its homologue, OXR1, it is capable of reducing the DNA damaging effects of reactive oxygen species when expressed in bacteria, indicating the protein has an activity that can contribute to oxidation resistance. Unlike OXR1, it appears to localize to nuclei and interacts with the estrogen receptor. This raises the possibility that NCOA7 encodes the nuclear counterpart of the mitochondrial OXR1 protein and in mammalian cells it may reduce the oxidative by-products of estrogen metabolite-mediated DNA damage.

Mutations in DNA polymerase eta are not detected in squamous cell carcinoma of the skin.

The major etiological agent in skin cancer is exposure to UV-irradiation and the concomitant DNA damage. UV-induced DNA lesions, such as thymine dimers, block DNA synthesis by the major DNA polymerases and inhibit the progression of DNA replication. Bypass of thymine dimers and related lesions is dependent on the translesion polymerase DNA polymerase eta (Poleta). In the inherited disorder, xeroderma pigmentosum variant (XPV), inactivation of Poleta results in extreme sensitivity to UV light and a marked increase in the incidence of skin cancer. Here, we tested the hypothesis that somatic mutations and/or polymorphisms in the POLH gene that encodes Poleta are associated with the induction of UV-dependent skin cancers. We sequenced the exonic regions of the Poleta open reading frame in DNA from 17 paired samples of squamous cell skin carcinoma and adjacent histologically normal tissue. We analyzed approximately 120,000 nucleotides and detected no mutations in POLH in the tumors. However, we identified 6 different single-nucleotide polymorphisms, 3 of them previously undocumented, which were present in both the tumor and paired normal tissue. We conclude that neither mutations nor polymorphisms in the coding regions of POLH are required for the generation of human skin squamous cell carcinoma.

Stress induction and mitochondrial localization of Oxr1 proteins in yeast and humans.

Reactive oxygen species (ROS) are critical molecules produced as a consequence of aerobic respiration. It is essential for cells to control the production and activity of such molecules in order to protect the genome and regulate cellular processes such as stress response and apoptosis. Mitochondria are the major source of ROS within the cell, and as a result, numerous proteins have evolved to prevent or repair oxidative damage in this organelle. The recently discovered OXR1 gene family represents a set of conserved eukaryotic genes. Previous studies of the yeast OXR1 gene indicate that it functions to protect cells from oxidative damage. In this report, we show that human and yeast OXR1 genes are induced by heat and oxidative stress and that their proteins localize to the mitochondria and function to protect against oxidative damage. We also demonstrate that mitochondrial localization is required for Oxr1 protein to prevent oxidative damage.