Quantitative single-molecule FRET and its application in experiments on DNA damage recognition by PARP-1

Abstract

Single-molecule Förster resonance energy transfer (smFRET) is a popular tool for characterizing biomolecules and investigating biological processes, as it can monitor distances on the nanometer scale. Most laboratories use their own custom-built setups and data analysis protocols for such studies. In view of these experimental differences, it is important to establish standard procedures which yield reproducible results. The first part of this thesis gives an introduction to the analysis of confocal smFRET data. Different strategies for determining correction factors are presented and discussed using a set of reference samples. The extracted FRET efficiencies are compared to theoretical values and experimental means from other labs. Experimental uncertainties are evaluated and discussed. Additionally, a short comparison of a dynamic species and a mixture of static molecules is presented. In the second part of this thesis, an smFRET assay is used for investigating how the nuclear enzyme poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA single-strand breaks. Two N-terminal zinc fingers of the protein are of special interest, as they are crucial for damage recognition. They are known to bind damaged DNA in a kinked conformation. The role of these two zinc fingers in DNA binding is further investigated by monitoring the DNA conformation upon protein binding via smFRET. A combination of quantitative smFRET results with molecular modeling and simulations identifies possible DNA conformations in complex with the zinc fingers and the respective kinking angles are analyzed