Ph.D., Biochemistry, University of California, Berkeley
B.A., Biology, Wesleyan University
Our research engages a range of topics at the intersection of biomedicine and the environment. This interest has been driven by trying to understand how cells defend against damaging molecules that are endogenous to cells but also produced by environmental agents. One strong theme has been defining the biochemical and biological functions of repair pathways for oxidative DNA damage. Another major theme of our work has been discovering and defining genetic regulatory systems that govern cellular responses to oxidative stress and nitric oxide. These two areas synergize in various ways: for example, the oxidative stress caused by arsenite exposure activates the APE1 gene, which encodes a central component of DNA repair and is vital to cell survival. Some recent studies expanded our analysis to mitochondria, the cellular energy organelles that maintain their own DNA genome. DNA repair in mitochondria was considered very limited, but these studies have demonstrated new proteins and pathways that were previously thought lacking. Investigations continue on Ape1 protein functions in the cell beyond DNA repair, and on understanding the mechanisms that partition oxidative DNA lesions among competing repair and mutational pathways.
Prof. Demple has been involved in research on DNA repair since his graduate studies at U.C. Berkeley, where he discovered the first DNA glycosylase acting on oxidative DNA damage. As a postdoctoral fellow, he isolated the sacrificial repair protein O6-methylguanine-DNA methyltransferase and showed that it acts with “suicide” kinetics. He identified the active site cysteine, and used protein sequence information to help clone the ada gene encoding the methyltransferase. During this time, he also carried out independent work establishing the existence of an oxidative stress response to hydrogen peroxide. Prof. Demple began his own laboratory in 1984 with studies of both DNA repair and cellular responses to oxidative stress, and the group has pursued aspects of both ever since. Some key discoveries include:
1) indentifying the enzymes in bacteria, yeast and human cells that excise 3’-fragments of deoxyribose at oxidative strand breaks, and cloning the yeast (APN1) and human (APE1) genes encoding these enzymes;
2) discovery of the oxyR-dependent control of metabolic H2O2 production in E. coli;
3) discovery of the soxRS regulatory system that responds to stress by superoxide-generating agents;
4) defined the redox activation mechanism of SoxR dependent on its iron-sulfur centers;
5) demonstrated the soxRS role in resistance to macrophages that generate nitric oxide;
6) showed that soxR mutations aid the development of some clinical antibiotic resistance;
7) defined the mutational specificity of abasic sites generated endogenously in yeast;
8) demonstrated that the DNA repair activity of Ape1 protein is essential, even at the level of individual cells;
9) showed that the oxidative lesion 2-deoxyribonolactone forms mechanism-based DNA-protein crosslinks with the repair DNA polymerase b, and that such crosslinking can be avoided by the activation of long-patch base excision DNA repair;
10) discovered that long-patch base excision repair occurs in mitochondria, where it depends on two proteins thought to be only in the nucleus, the structure-specific endonucleases FEN1 and Dna2.
1. Demple, B., Johnson, A., and Fung, D. (1986) Exonuclease III and endonuclease IV remove 3' blocks from DNA synthesis primers in H2O2-damaged Escherichia coli, Proc Natl Acad Sci U S A 83, 7731-7735.
2. Johnson, A. W., and Demple, B. (1988) Yeast DNA diesterase for 3'-fragments of deoxyribose: purification and physical properties of a repair enzyme for oxidative DNA damage, J Biol Chem 263, 18009-18016.
3. Greenberg, J. T., Monach, P., Chou, J. H., Josephy, P. D., and Demple, B. (1990) Positive control of a global antioxidant defense regulon activated by superoxide-generating agents in Escherichia coli, Proc Natl Acad Sci U S A 87, 6181-6185.
4. Amabile-Cuevas, C. F., and Demple, B. (1991) Molecular characterization of the soxRS genes of Escherichia coli: two genes control a superoxide stress regulon, Nucleic Acids Research 19, 4479-4484.
5. Demple, B., Herman, T., and Chen, D. S. (1991) Cloning and expression of APE, the cDNA encoding the major human apurinic endonuclease: definition of a family of DNA repair enzymes, Proc Natl Acad Sci U S A 88, 11450-11454.
6. Nunoshiba, T., deRojas-Walker, T., Wishnok, J. S., Tannenbaum, S. R., and Demple, B. (1993) Activation by nitric oxide of an oxidative-stress response that defends Escherichia coli against activated macrophages, Proc Natl Acad Sci U S A 90, 9993-9997.
7. Hidalgo, E., and Demple, B. (1994) An iron-sulfur center essential for transcriptional activation by the redox-sensing SoxR protein, EMBO Journal 13, 138-146.
8. Ding, H., Hidalgo, E., and Demple, B. (1996) The redox state of the [2Fe-2S] clusters in SoxR protein regulates its activity as a transcription factor, Journal of Biological Chemistry 271, 33173-33175.
9. Bennett, R. A. O., Wilson, D. M. III, Wong, D., and Demple, B. (1997) Interaction of human apurinic endonuclease and DNA polymerase beta in the base excision repair pathway, Proc Natl Acad Sci U S A 94, 7166-7169.
10. Hidalgo, E., Ding, H., and Demple, B. (1997) Redox signal transduction: mutations shifting [2Fe-2S] centers of the SoxR sensor-regulator to the oxidized form, Cell 88, 121-129.
11. Marquis, J. C., and Demple, B. (1998) Complex genetic response of human cells to sublethal levels of pure nitric oxide, Cancer Res 58, 3435-3440.
12. DeMott, M. S., Beyret, E., Wong, D., Bales, B. C., Hwang, J. T., Greenberg, M. M., and Demple, B. (2002) Covalent trapping of human DNA polymerase beta by the oxidative DNA lesion 2-deoxyribonolactone, J Biol Chem 277, 7637-7640.
13. Wong, D., and Demple, B. (2004) Modulation of the 5'-deoxyribose-5-phosphate lyase and DNA synthesis activities of mammalian DNA polymerase beta by apurinic/apyrimidinic endonuclease 1, J Biol Chem 279, 25268-25275.
14. Auerbach, P., Bennett, R. A., Bailey, E. A., Krokan, H. E., and Demple, B. (2005) Mutagenic specificity of endogenously generated abasic sites in Saccharomyces cerevisiae chromosomal DNA, Proc Natl Acad Sci U S A 102, 17711-17716.
15. Fung, H., and Demple, B. (2005) A vital role for Ape1/Ref1 protein in repairing spontaneous DNA damage in human cells, Mol Cell 17, 463-470.
16. Koutsolioutsou, A., Pena-Llopis, S., and Demple, B. (2005) Constitutive soxR mutations contribute to multiple-antibiotic resistance in clinical Escherichia coli isolates, Antimicrob Agents Chemother 49, 2746-2752.
17. McLaughlin, L. M., and Demple, B. (2005) Nitric oxide-induced apoptosis in lymphoblastoid and fibroblast cells dependent on the phosphorylation and activation of p53, Cancer Res 65, 6097-6104.
18. Sung, J. S., DeMott, M. S., and Demple, B. (2005) Long-patch base excision DNA repair of 2-deoxyribonolactone prevents the formation of DNA-protein cross-links with DNA polymerase beta, J Biol Chem 280, 39095-39103.
19. Fung, H., Liu, P., and Demple, B. (2007) ATF4-dependent oxidative induction of the DNA repair enzyme Ape1 counteracts arsenite cytotoxicity and suppresses arsenite-mediated mutagenesis, Molecular and Cellular Biology 27, 8834-8847.
20. Liu, P., Qian, L., Sung, J. S., de Souza-Pinto, N. C., Zheng, L., Bogenhagen, D. F., Bohr, V. A., Wilson, D. M., 3rd, Shen, B., and Demple, B. (2008) Removal of oxidative DNA damage via FEN1-dependent long-patch base excision repair in human cell mitochondria, Molecular and Cellular Biology 28, 4975-4987.
21. Fung, H., and Demple, B. (2011) Distinct roles of Ape1 protein in the repair of DNA damage induced by ionizing radiation or bleomycin, J Biol Chem 286, 4968-4977.
22. Madlener, S., Ströbel, T., Vose, S., Saydam, O., Price, B.D., Demple, B., and Saydam, N. (2013) An essential role for mammalian AP endonuclease Ape1/Ref-1 in telomere maintenance, Proceedings of the National Academy of Sciences USA 110, 17844-17849.
23. Quiñones, J.L., Thapar, U., Yu, K., Fang, Q., Sobol, R.W., and Demple, B. (2015) Enzyme Mechanism-Based, Oxidative DNA-Protein Crosslinks Formed with DNA Polymerase β in vivo, Proceedings of the National Academy of Sciences of the USA 112, 8602-8607.