New light on the AGT DNA alkyltransferase lesion repair mechanism with Graphene Energy Transfer on vertical Nucleic Acids (GETvNA)

• Applicants: Ingrid Teßmer, Ph.D.; Professor Dr. Philip Tinnefeld
• Subject Area: Biophysics ; Biochemistry
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 550215301

The O6-alkylguanine DNA alkyltransferase, AGT is responsible for removing highly mutagenic and cytotoxic alkyl lesions from DNA and is thus an important player in cell survival by maintaining genome integrity. In addition, AGT has also become a focus of targeted inhibitor development, because its repair activity counters the toxic effects of alkylation damage that is deliberately introduced into DNA by certain types of cancer chemotherapeutic agents. A greater understanding of the mechanism of DNA lesion repair by AGT therefore has the potential to be highly beneficial in both the prevention and the treatment of human cancer. We have recently developed a powerful, new methodology based on single molecule fluorescence quenching through graphene induced energy transfer (graphene-induced energy transfer with vertical nucleic acids, GETvNA). Due to the steep distance dependence of the energy transfer, GETvNA provides unprecedented spatial resolution in the sub-Ångstrom range of fluorescently labelled proteins on DNA substrates that are vertically attached on a graphene surface, al-lowing us to access the short distance regime of DNA translocations and DNA lesion interactions by AGT with high temporal resolution. We plan to apply this method combined with complementary techniques such as MINFLUX, AFM, as well as cellular assays to provide mechanistic understanding of AGT-DNA and AGT-lesion interactions. In particular, we will focus on the role of the previously observed cooperative cluster formation by AGT in lesion detection and processing in vitro and in vivo.

• DFG Programme: Research Grants