Research Interests:

Oxygen-derived free radicals occur as spontaneous metabolic by-products in aerobic organisms, or at high levels as cytotoxic weapons of the immune system. Nitric oxide (NO) is another free radical with dual roles: at low levels in intercellular signaling, and at high levels during inflammatory responses. Our extensive work with the important model SoxR protein of E. coli has shown it to be an unusual transcription factor with [2Fe-2S] clusters: SoxR transcriptional activity is triggered by oxidation of the metal centers, or by their reaction with NO to form mixed iron-nitrosyl complexes. The activation of SoxR induces the expression of dozens of genes that contribute to bacterial resistance to oxidants, some heavy metals, and organic solvents. The SoxR-regulated system also confers broad antibiotic resistance; indeed, mutational activation of SoxR has been detected in clinical infections displaying resistance to quinolone antibiotics.

A major interest in our group concerns the mechanisms and roles of human enzymes that repair oxidative (free radical) damage in DNA. We have focused on the main pathway, base excision DNA repair (BER), which handles most of the oxidative DNA damage from both endogenous and environmental sources. Pivotal in this process is Ape1, the abasic endonuclease that channels most damage into a central BER pathway. The DNA repair of Ape1 is essential for cell viability, and Ape1 expression is regulated dynamically at the level of transcription. The nature of the signaling in this response, and the exact role(s) played by Ape1 as part of it, remain important questions. The source(s) of the endogenous DNA damage that lead to the vital requirement for Ape1 also need to be defined. Our work has also identified the importance of a class of oxidative damage that had been ignored in the past: oxidized abasic sites. Among these, the lesion 2-deoxyribonolactone (deoxyribose oxidized at the C1 position) is especially dangerous. Ape1 cleaves 2-deoxyribonolactone effectively, but the next enzyme in the BER pathway, DNA polymerase ß, becomes stuck in an irreversible covalent complex with the DNA when attempting the next stage of repair. We have shown that, in the nucleus, most of the problem can be avoided by activating a branch of BER in which the activities of polymerase ß are supplanted by replication enzymes. Recent work has extended this discovery to mitochondrial DNA repair, where DNA polymerase y, the only replication and repair enzyme, has the same risk of crosslinking as seen for polymerase ß. We showed that the “flap endonuclease” FEN1, thought to be absent from mitochondria, is actually present there and participates in repairing such dangerous lesions in DNA. The mechanism of the switching process that allows repair to proceed is a key unknown that we wish to address.

NO also activates resistance pathways in human cells, but the regulatory proteins remain to be identified. We have shown that increased NO resistance in mammalian cells involves the inducible enzyme heme oxygenase 1, and this induction is controlled by dramatically increased stability of the corresponding mRNA. We have now identified the regulator (HuR) that is activated by NO to stabilize the heme oxygenase mRNA, which opens the door to defining the signaling process in molecular terms.

Selected Publications:

M. Chander and B. Demple. 2004. Functional analysis of SoxR residues necessary for transducing stress signals into gene expression. J. Biol. Chem. 279: 41603-41610.

H. Fung and B. Demple. 2005. Vital Role of Ape1/Ref1 Protein in Repairing Spontaneous DNA Damage in Human Cells. Molecular Cell 17: 463-470.

Hua Fung, Pingfang Liu and Bruce Demple. 2007. ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-mediated Mutagenesis. Mol. Cell. Biol. 27: 8834-8847.

Pingfang Liu, Limin Qian, Jung-Suk Sung, Nadja C. de Souza-Pinto, Li Zheng, Daniel F. Bogenhagen, Vilhelm A. Bohr, David M. Wilson III, Binghui Shen and Bruce Demple. 2008. Removal of Oxidative DNA Damage via FEN1-Dependent Long-Patch Base Excision Repair in Human Cell Mitochondria. Mol. Cell. Biol. 28: 4975-4987.

L.M. McLaughlin and B. Demple. 2005. Nitric oxide-induced apoptosis in lymphoblastoid and fibroblast cells dependent on the phosphorylation and activation of p53. Cancer Research 65: 6097-6104.

Veronica Leautaud and Bruce Demple. 2007. Regulation of heme oxygenase-1 mRNA deadenylation and turnover in NIH3T3 cells during alkylation or nitrosative stress. BMC Molecular Biology 8: 116 (open source).