A new single molecule approach to study DNA repair protein dynamics: seeing is believing Instabilité Génétique et Cancer

This seminar will present a new rapid and robust method for single molecule analysis of DNA binding proteins from nuclear extracts (SMADNE) of human cells expressing a fluorescently tagged protein of interest (Schaich et al., Nucleic Acids Res. 2023 Apr 24;51(7):e39). SMADNE when combined with the LUMICKS C-trap provides unprecedented observations of DNA repair protein dynamics on damaged DNA. Over the last 18 months our group has been able to analyze the dwell times of 30 proteins or protein variants on DNA substrates using this approach. After a brief introduction to DNA damage and base excision repair, this seminar will discuss new unpublished data, including the DNA binding dynamics of PARP1 and PARP1 variants or DNA LIG3-XRCC1 on nicks in naked DNA and in a nucleosome. An important glycosylase, TDG, involved in active oxidative demethylation of 5mC through base excision repair will also be discussed. Finally, the non-specific DNA interactions and the binding behavior to 8-oxoG of purified eGFP-OGG1, purified eGFP-OGG1 added to extracts, and eGFP-OGG1 from human cell nuclear extracts will be directly compared.