Our work centers on the mechanisms by which diverse organisms (bacteria, yeast, and human cells) deal with the toxic side effects of free radicals. Aerobic cells use oxygen for energy production, but oxygen also generates free radicals that, if they are not kept in check, can cause cellular and genetic damage. The immune system actively generates free radicals such as superoxide and nitric oxide as weapons against pathogens and tumor cells, but normal cells nearby may also be damaged. The defenses against free radical damage are remarkable. They include specialized repair enzymes that correct oxidative damages in DNA, and detoxification systems such as superoxide dismutase. In many organisms, these defenses are coordinated genetically as global responses.

Our work comprises three main areas:

1. DNA Repair
2. Adaptive Resistance to Nitric Oxide
3. The soxRS Regulon

 

1. DNA Repair

We are defining the biochemical and biological roles of eukaryotic DNA repair enzymes that initiate the correction of sites where whole nucleotide fragments have been eliminated by free radical reactions; these lesions can lead to mutations if they are not repaired. We have shown that the human Ape1 protein plays a pivotal role in correcting diverse types of free radical damage to DNA, with its activity coupled to other proteins in the same repair pathway. We have also investigated how the expression of Ape1 is controlled, and find that it is coupled to both the cell cycle to oxidative stress. Recent work demonstrates that Ape1 plays an essential role in handling DNA damage generated by normal metabolism, and we are investigating the biological sources of this "spontaneous" DNA damage.

Related Publications:

Yang H, Clendenin WM, Wong D, Demple B, Slupska MM, Chiang JH and Miller JH. Enhanced Activity of Adenine-DNA Glycosylase (Myh) by Apurinic/apyrimidinic endonuclease (Ape1) in Mammalian Base Excision Repair of an A/GO Mismatch. Nucleic Acids Research, 29:743-752, 2001.

Fung H, Bennett RAO, and DEMPLE B. Key role of a downstream Sp1 site in cell-cycle regulated transcription of the AP endonuclease gene APE1/APEX in NIH3T3 cells. J Biol Chem, 276: 42011-42017, 2001.

DeMott MS, Beyret E, Wong D, Bales BC, Hwang JT, Greenberg MM, and DEMPLE B. Covalent Trapping of Human DNA Polymerase b by the Oxidative DNA Lesion 2-Deoxyribonolactone. J Biol Chem: 7637-7640, 2002.

Xu YJ, DeMott MS, Hwang JT, Greenberg MM, and DEMPLE B. Action of Human Apurinic Endonuclease (Ape1) on C1’-Oxidized Deoxyribose Damage in DNA. DNA Repair, 108:1-11, 2003.

DEMPLE B and DeMott MS. Dynamics and Diversions in Base Excision DNA Repair of Oxidized Abasic Lesions. Oncogene Reviews (ed. M. Sekiguchi) 21: 8926-8934, 2002.

Wong D, DeMott MS, and DEMPLE B. Modulation of the 3’->5’ Exonuclease Activity of Human Apurinic Endonuclease (Ape1) by Its 5’-incised Abasic DNA Product. J Biol Chem 278: 36242-36249, 2003.

D. Wong and B. DEMPLE. 2004. Modulation of the 5'-deoxyribose-5-phosphate lyase and DNA synthesis activities of mammalian DNA polymerase beta by AP endonuclease-1. Journal of Biological Chemistry 279: 25268-25275.

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.

2. Adaptive Resistance to Nitric Oxide

We have described how human cells modulate resistance to the free radical nitric oxide by varying the expression of many defense genes in a process termed adaptive resistance. Key among the inducible functions is heme oxygenase 1, and we have shown that nitric oxide induces its expression by uniquely stabilizing the heme oxygenase 1 mRNA. We are investigating the mechanism of this new signal transduction pathway, and the identity and role of various genes in adaptive resistance to nitric oxide.

Related Publications:

Marquis JC and Demple B. Complex Genetic Response of Human Cells to Sublethal Levels of Pure Nitric Oxide. Cancer Res, 58:3435-3440, 1998.

Bishop A, Marquis JC, Cashman NR and Demple B. Adaptive Resistance to Nitric Oxide in Motor Neurons. Free Rad Biol Med. 26:978-986, 1999.

Bouton C and Demple B. Nitric oxide-inducible expression of heme oxygenase-1 in human cells: Translation-independent stabilization of the mRNA and evidence for direct action of NO. Journal of Biological Chemistry 275:32688-32693, 2000.

DEMPLE B. Signal Transduction by Nitric Oxide in Cellular Stress Responses. Molecular and Cellular Biochemistry 234/235: 11-18, 2002.

Mitsumoto M, Mitsumoto A, and DEMPLE B. Nitric oxide-mediated upregulation of the TGFb-inducible early response gene-1 (TIEG1) in human fibroblasts by mRNA stabilization independent of TGFb. Free Radical Biology and Medicine, 34(12):1607-1613, 2003.

A. Bishop, S. Fung-Yet, M.J. Perrella, A.M. Lee, N.R. Cashman and B. DEMPLE. 2004. A Role for Heme Oxygenase-1 in Nitric Oxide Resistance in murine motor neurons. Biochemical and Biophysical Research Communications 325: 5-9.

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

3. The soxRS Regulon

We have defined an important regulatory system in E. coli that governs a complex of genes, the soxRS regulon, which is activated by increased superoxide production in the cell. This system is also triggered by the nitric oxide that some immune cells generate to defeat pathogens. A master protein, called SoxR, is the key regulatory protein in this system. SoxR is a novel transcription factor containing a redox-active iron-sulfur center. We are investigating how reactions with free radicals modulate SoxR activity, and the mechanism by which the activated protein switches on transcription. Activation of the soxRS regulon also increases bacterial resistance to diverse antibiotics. We are investigating how this chromosomally-mediated antibiotic resistance contributes to the spread of resistance in clinical infections of Salmonella and other pathogens.

Related Publications:

Hidalgo E, Ding H and Demple B. Redox signal transduction: mutations shifting [2 Fe-2S] Centers of the SoxR sensor-regulator to the oxidized form. Cell, 88:121-129, 1997.

Pomposiello P and Demple B. Identification of SoxS-Regulated Genes in Salmonella enterica Serovar Typhimurium. J Bacteriol, 182:23-29, 2000.

Ding H and Demple B. Direct Nitric Oxide Signal Transduction via Nitrosylation of Iron-Sulfur Centers in the SoxR Transcription Factor. Proc. Natl. Acad. Sci. USA, 97:5146-5150, 2000.

Kwon HJ, Bennik MHJ, Demple B and Ellenberger T. Crystal Structure of the Escherichia coli Rob Transcription Factor in Complex with DNA. Nature Structural Biology, 7:424-430, 2000.

Koutsolioutsou A, Martins EA, White DG, Levy SB and Demple B. A soxRS-Constitutive Mutation Contributing to Antibiotic Resistance in a Clinical Isolate of Salmonella enterica (Serovar Typhimurium). Antimicrobial Agents and Chemotherapy, 45:38-45, 2001.

Pomposiello P and Demple B. Redox-operated genetic switches: The SoxR and OxyR Transcription Factors. Trends in Biotechnology, 19:109-114, 2001.

Chander M, Raducha-Grace L, and DEMPLE B. Transcription-defective soxR mutants of Escherichia coli: Isolation and in vivo characterization. J. Bacteriol., 185:2441-2450, 2003.

M. Chander and B. DEMPLE. 2004. Functional analysis of SoxR residues necessary for transducing stress signals into gene expression. Journal of Biological Chemistry 279: 41603-41610.

A. Koutsolioutsou, S. Peña-Llopis and B. DEMPLE. 2005. Constitutive soxR Mutations Contribute to Multiple Antibiotic Resistance in Clinical Escherichia coli Isolates. Antimicrobial Agents and Chemotherapy 49: 2746-2752.

Ph.D., 1981, University of California, Berkeley