Research in the Georgiadis lab focuses on understanding the mechanisms by which protein-nucleic acid interactions regulate biological processes including transcription, replication, and DNA repair. Investigation of these functionally significant protein-nucleic acid interactions involves structural work, primarily X-ray crystallography, NMR, and cryoEM, complementary biochemistry and/or enzymology, development of small molecule probes, and cell-based approaches.
Conformational dynamics of Apurinic/apyrimidinic endonuclease (APE1)
The Georgiadis laboratory has a long-standing interest in APE1, a protein that serves as an essential base excision repair enzyme and as a redox factor. DNA repair enzymes are up-regulated in many cancers and have recently been the focus of cancer therapeutic development. Among the DNA repair enzymes, APE1 is unique in having a dual role as an enzyme and a redox factor; both of these functions have been targeted for drug development by academic laboratories and companies. From a structural perspective, the most intriguing feature of the dual functional roles is that they almost certainly involve conformationally distinct forms of the enzyme. Early evidence for multiple conformational states of APE1 was achieved in collaboration with Mike Gross (Wash U) through the development of an NEM-footprinting assay (Su et al, Biochemistry, 2011). Recent work has shifted to approaches that will detail the nature of conformational dynamics in APE1, specifically NMR approaches made possible by the recent acquisition of a Bruker 600 MHz instrument in the Chemical Genomics Core facility. In collaboration with Ratan Rai (IUSM), the Georgiadis laboratory is exploring dynamics in APE1 and the impact of small molecule inhibitors on that motion. Georgiadis lab is also pursuing development of novel chemical probes targeting APE1.
Alien DNA as an alternate genetic system
In a collaborative project with Steven Benner (Foundation for Applied Molecular Evolution), Georgiadis laboratory is investigating the structural properties of Alien DNA (Alien in the sense that it is not natural DNA) and the ability of DNA polymerases to recognize and faithfully replicate this unnatural DNA. Benner lab has spent the past 25 years developing unnatural nucleobase pairs that take advantage of orthogonal hydrogen-bonding interactions to create an expanded genetic information system and most recently entirely unnatural or Alien DNA. Initial work on this project focused on a 6-letter alphabet including A-T, G-C, and P-Z pairs (Georgiadis et al, JACS, 2015; Molt et al, NAR, 2017). Georgiadis lab provided insights into the recognition of 6-letter DNA by an evolved DNA polymerase in crystal structures of pre- and post-incorporation complexes (Singh, NAR, 2018). The project then expanded to hachimoji DNA, 8-letter DNA, including A-T, G-C, P-Z, and B-S pairs (Hoshika et al. Science, 2019). The work is now focused on Alien DNA including P-Z and B-S pairs. Georgiadis lab determined high-resolution crystal structures of Alien DNA in both A- and B-form. Efforts to obtain complexes of Alien DNA with evolved DNA polymerases are in progress.
The role of SETMAR in bladder cancer
The Georgiadis lab has for several years been interested in characterizing the function of a sequence-specific DNA-binding protein, SETMAR. This protein arose as a chimeric fusion in simian primates following insertion of a DNA transposon, Hsmar1, in the primate lineage. The domesticated transposase domain from Hrmar1 was fused to a SET (lysine methyltransferase) domain creating SETMAR. Initial work focused on structural characterization of the transposase catalytic domain(Goodwin et al, Biochemistry 2010) and a complex of the DNA-binding domain bound to the Hsmar1 terminal inverted repeat DNA (Chen et al, 2022, JBC). A recent report in the literature (Xie et al, 2021, Mol Ther), suggests that alternative splicing of SETMAR plays a role in metastasis in bladder cancer; the full-length protein including the SET domain is protective while a splice variant lacking the SET domain is detrimental. In collaboration with Evan Cornett (IUSM), studies are now focused on substrate specificity of the SET domain and the role of sequence-specific DNA binding activity associated with the transposase domain in bladder cancer.
Other projects include a collaborative project with Michael Weiss, MD, PhD on the male specific transcription factor SRY.
Current research funding
Investigation of novel signaling protein in 3D and in vivo PDAC models using second-generation Ref-1 inhibitors.
R01 CA254110-01A1 (PIs: Fishel/Han, Co-Is Georgiadis/Kelley)
Funding period: 06/01/21–5/31/26
Darwinism from Artificial Genomes
1939086 (PIs: Benner/Georgiadis)
Funding period: 12/15/19–11/30/23
Mechanistic Role of APE1 and BER in chemotherapy-induced peripheral neurophathy
R01 CA205166-01A1 (MPI:Kelley/Fehrenbacher, Co-I Georgiadis)
Funding period: 04/01/17–02/28/23
Regulation of RNA Polymerase II Transcription by the Phosphatase Rtr1
2R01GM099714-06 CR (PI: Mosley, Co-I Georgiadis)
NIH / NIGMS
Funding period: 08/01/17–07/31/22
Hoshika, S., Leal, N.A., Kim, M.J., Kim, M.S., Karalkar, N.B., Kim, J.G., Bates, A.M., Watkins, N.E., Jr., SantaLucia, H.A., Meyer, A.J., DasGupta, S., Piccirilli, J.A. Ellington, A.D. SantaLucia, J., Jr., Georgiadis, M.M., Benner, S.A. (2019) Hachimoji DNA and RNA: A genetic system with eight building blocks. Science 363, 884-887. PMCID: PMC6413494 DOI: 10.1126/science.aat0971
Ouaray, Z., Benner, S.A., Georgiadis, M.M., and Richards, N.G.J. (2020) Building better polymerases: Engineering the replication of expanded genetic alphabets. J Biol Chem 295, 17046-17059. PMID: 33453957 DOI: 10.1074/jbc.REV120.013745
Wilson, D.M., III, Deacon, A.M., Duncton, M.A.J., Pellicena, P., Georgiadis, M.M., Yeh, A.P., Arvai, A.S., Moiani, D., Tainer, J.A., and Das, D. (2020) Fragment- and structure-based drug discovery for developing therapeutic agents targeting the DNA Damage Response. Prog Biophys Mol Biol, S0079-6107(20)30111-5. PMID: 33115610 DOI: 10.1016/j.pbiomolbio.2020.10.005
Paavola, J.L., Battistin, U., Ogata, C.M., and Georgiadis, M.M. (2021) Crystal structures of a dodecameric multicopper oxidase from Marinithermus hydrothermalis. Acta Cryst D 77, 1336-1345 DOI: 10.1107/S205979832100944X
Chen, Q., Bates, A.M., Hanquier, J. N., Simpson, E., Rusch, D. B., Podichit, R., Liu, Y., Wek, R.C., Cornett, E. M., and Georgiadis, M.M. (2022) Structure and genome-wide anlysses suggest that transposon-derived portein SETMAR alters transcription and splicing. J. Biol. Chem. 298, DOI: 10.1016/j.jbc.2022.101894
See a complete list of publications from the Georgiadis Lab in PubMed.
Erin Smith, BS