Georgiadis Lab

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. 

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 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.