Structural and Biophysical Studies of non-canonical DNA
This talk will cover two representative stories on our attempts to elucidate the structure of non-canonical DNA implicated in important biological processes. To accomplish this goal we used a variety of biophysical methods coupled with X-ray crystallography. The first project concerns (CAGAGG)n repeats identified in difficult-to-replicate sequences in the mouse genome. Such repeats promote the collapse of DNA replication forks contributing to the breakpoints and translocations that cause cancer. We hypothesize that (CAGAGG)n repeat stalls the replication fork because it adopts a stable secondary structure. In order to elucidate its nature, we investigated the structure and stability of (CAGAGG)n repeats with n = 2-15 as well as their variants and mutants using circular dichroism, UV-vis and fluorescence spectroscopy, gel electrophoresis, thermal stability, mass spectrometry, and analytical ultracentrifugation. X-ray crystallography was used to investigate the atomic details of (CAGAGG)n structure. Based on the summary of our results we propose a tetra-stranded model, which we termed 1Core, that includes canonical Watson-Crick G-C base-pairs in the core and non-canonical G-G and G-A pairing in the loops (figure on the left).
In the second part of my talk, I will discuss our structural and biophysical characterization of a variety of telomeric and oncogene promoter DNA sequences (e.g. human telomeric DNA (Tel22); telomeric DNA from Tetrahymena thermofila (THM); human c-Myc oncogene promoter) in complex with N-methylmesoporphyrin IX (NMM) and other GQ ligands. Due to excellent optical properties of NMM, we investigated its binding to GQs using UV-vis and fluorescence titrations. We determined stoichiometries for binding events independently via the Job plot method. We obtained thermodynamic parameters via ITC titrations. We crystallized NMM in complex with Tel22 and THM (figure on the right). Our findings are of great importance for the development of new and efficient anticancer therapies.
Chemistry and Biochemistry, Swarthmore College
Domain 1 - UMR 9187 / U1196 - Chemistry, Modelling and Imaging for Biology