The cellular genome is continually exposed to hazards that damage the DNA and reduce its stability, consequently triggering deleterious human health outcomes. Importantly, multiple enzymes involved in maintaining genomic stability are deregulated or mutated in cancer cells. While a general connection between DNA damage and human health has been established, it remains unclear how genome stability is altered at the molecular level. In this sense, the "devil is truly in the details" and gaining mechanistic insight is essential.
EMGS Young Scientist Award Video
The overarching goal of the lab is to fill the knowledge gap between DNA damage and human disease in hopes of beneficially impacting the treatment and/or prevention of human ailments. We are particularly interested in oxidative DNA damage because the basis of multiple human afflictions are rooted in oxidative stress. A primary defense mechanism employed during the repair of oxidative DNA damage is Base Excision Repair (BER). BER involves the removal of the damaged base and subsequent processing by a multi-protein complex that protects the cell from toxic DNA intermediates.
Major areas of research within the lab include:
1. Understanding the DNA polymerase mechanism at the molecular level
While the basic DNA polymerase mechanism has been established for non-damaged DNA, it has recently come to light that not all DNA damage is handled the same way. This complicates the mechanistic and biological interpretation of how DNA polymerases handle DNA damage, therapeutic nucleotides, and other modified bases. To address this, we mechanistically characterize how DNA polymerases process DNA lesions using time-lapse crystallography, neutron crystallography, pre-steady state kinetics, equilibrium binding studies, and mutational analysis. The lab has employed multiple mammalian DNA polymerases involved in both base excision repair (pol beta, shown below in green) and translesion synthesis (rev1, shown below in white, and pol kappa) to study the DNA polymerase mechanism.
2. Determining the mechanism of APE1 during repair of DNA damage
AP-Endonuclease 1 (APE1) is a complex DNA repair protein involved in BER, nucleotide incision repair (NIR), and RNA processing. These biological processes are mediated mainly through APE1 phosphodiester backbone cleavage reactions. We are specifically interested in the molecular mechanisms used by APE1 to cleanse damaged dirty ends that block replication/repair and how APE1 processes a wide array of DNA damage. By addressing these questions, the laboratory has made significant contributions toward understanding the mechanism of APE1.
3. Elucidating the dynamics of the base excision repair (BER) complex during the course of DNA repair
Specifically, we are interested in how BER complexes are formed and channel toxic DNA intermediates between each protein during the course of repair. To address dynamics of the BER complex, we have custom built a three-color single-molecule total internal reflection microscope (TIRFM) to characterize the dynamic assembly and disassembly of BER complexes. The system is fully functional and we are characterizing substrate channeling between each BER protein during repair of oxidative DNA damage. This approach has further diversified the labs experimental expertise and provides molecular insight into substrate channeling during BER.
4. Characterizing the telomerase catalytic mechanism
Our lab has determined novel X-ray crystal structures of the catalytic subunit of a human telomerase reverse transcriptase (TERT) homolog throughout the entire catalytic cycle. By combining structural snapshots with pre-steady-state kinetic analysis, we can assign mechanistic roles for key active site residues with regard to telomerase fidelity and ribonucleotide insertion. Importantly, we corroborate the trends we observe with our model TERT system with complementary studies with human telomerase, in collaboration with the O’presko lab (PITT). We aim to obtain a deeper understanding of how telomerase protects telomeric integrity by specifically selecting canonical rather than noncanonical nucleotides during telomeric insertion.
5. Identifying how BER proteins repair DNA damage within nucleosomes at the atomic and single molecule level
The overarching goal of this project is to understand how BER proteins repair DNA damage in a chromatin environment, where the DNA is packaged into nucleosomes. To accomplish this, we have established methodology to generate site-specific DNA damage within nucleosomes. Using these nucleosomes, we are able to study how BER proteins access and process DNA damage utilizing single-molecule fluorescence, enzyme kinetics, and cryo-EM. This is an exciting new project and builds on our lab's expertise in studying BER proteins.
6. Characterizing the anthrax toxin
This project utilizes cryo-EM to obtain structures of the functional lethal anthrax toxin. Moreover, the goal is to obtain complexes containing the N-terminal domain of lethal factor bound to the anthrax toxin protective antigen (PA) pore complexes inserted into lipid nanodiscs. The figure (left) below shows a cryoEM density map of the N-terminal domain of lethal factor translocating through protective antigen pore. We have already obtained multiple structures of this complex and our current working structure is at 2.97 Å. This project has resulted in the lab gaining extensive exposure to all things cryo-EM.
The lab utilizes a reductionist approach to investigate complex biological questions. To this end, we employ cutting edge biophysical approaches, which includes structural (X-ray, neutron, and cryo-EM), biochemical, kinetic, single-molecule, computational, and molecular biology assays. We have an in-house Rigaku MicroMax-007 HF rotating anode equipped with a Pilatus 200K detector that is utilized for the collection of publication quality macromolecular X-ray crystallographic data sets. Additionally, we have custom built a three-color single-molecule total internal reflection microscope (TIRFM). We are currently extending these approaches into cellular studies within our laboratory with collaborative guidance and expertise. This multi-technique approach ensures we have the tools necessary to answer pressing biological questions related to structural biology and DNA damage/repair.