DNA is the genetic material that gives each cell and organism its unique character. The accurate duplication of DNA as well as its subsequent transfer from parent to offspring is critical for life. Some environmental factors such as chemical carcinogens, UV light, or even natural cellular processes can cause oxidative or physical damage to our DNA, which can cause mutations or temporarily block its duplication. In fact, it has been estimated that there are tens of thousands of damaged DNA sites (lesions) in each human cell per day. Lesions in genomic DNA of other organisms are also widespread. If left unrepaired, these lesions can inhibit the duplication of DNA, cause cell death, or detrimental mutations. This project will use cutting-edge biophysical techniques to investigate how a commonly occurring oxidative DNA lesion prevents proteins from accurately duplicating the DNA. The results generated from this project will determine the consequences of DNA damage at the molecular level. In addition to its scientific importance, the project will also support undergraduate and graduate students at The Ohio State University with opportunities to receive scientific training, provide them tools for designing and testing scientific hypotheses, as well as learning about the nature of scientific discovery.
Cellular DNA is frequently damaged by both endogenous and exogenous sources to form a myriad of DNA damaged sites, which can stall the cellular DNA replication machinery during genome duplication. Some key proteins for DNA replication are the replicative DNA polymerases, which synthesize the majority of DNA, and translesion synthesis (TLS) DNA polymerases, which bypass and extend DNA across from damaged DNA sites but often in an error-prone way. The processivity factor PCNA enhances the ability of replicative polymerases to synthesize long stretches of DNA and plays a key role in polymerase switching at sites of DNA damage. It is not known how common DNA lesions affect the conformational dynamics of replicative and TLS polymerases in solution. Furthermore, it is not known how these two different types of polymerases are switched at a lesion site. To study the first mechanistic question, the project will utilize Förster resonance energy transfer techniques and a stopped-flow apparatus, a rapid mixer, to monitor the conformational dynamics of a replicative polymerase and a TLS polymerase during binding to substrates, or PCNA, as well as catalysis. To elucidate molecular details of polymerase switching, this project will employ cutting-edge single molecule techniques to investigate how a replicative polymerase and a TLS polymerase are switched on and off at a DNA damaged site with the help of PCNA. The project will also offer undergraduate and graduate students at The Ohio State University opportunities to receive important scientific training in the field of advanced enzymology. The project will provide them training in designing and testing scientific hypotheses as well as support for further career development and educational opportunities in STEM fields.
