Single-molecule studies of dynamic single stranded DNA damage recognition by PARP1

• Applicant: Professor Dr. Jens Michaelis
• Subject Area: Biophysics ; Biochemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 566094786

DNA damage repair, a complex intracellular pathway essential to all living organisms, starts with the recognition of the site of damage. Here, PARP1, which is the most abundant nuclear protein after histones, is crucial for recognizing the DNA damage, leading to a recruitment of the DNA repair machinery. The importance of this process is highlighted by the fact that PARP1 is the target for specific inhibitors used in cancer therapy. A mechanistic understanding of the underlying process is therefore extremely important. Here, we will use single-molecule FRET experiments to investigate the dynamics of PARP1 DNA damage recognition on well-defined DNA lesions such as single stranded nicks and gaps. Experiments will be performed in presence and absence of PARP inhibitors from different inhibitor classes to understand the mechanistic function of PARP inhibitors. Importantly we will investigate the dynamic interconversion between different structural states. Through a combination of quantitative smFRET measurements and structural modeling we aim to mechanistically decipher the recognition process. Experiments will also focus on the recruitment of XRCC1 to DNA damage sites by PARP1 and how the presence of XRCC1 alter the structure of the PARP1-DNA complex as well as its dynamics. In particular we will investigate the putative handoff between PARP1 and XRCC1. Recently, an important PARP1 interacting protein, HPF1 has become the focus of interest, since HPF1 alters the enzymatic activity of PARP1 in context of DNA damages. Here, we will therefore also investigate how HPF1 changes the dynamics of PARP1 DNA damage recognition using smFRET, as well as probe its effect on different PARP1 inhibitors. DNA damage inside of a eukaryotic cell has to be repaired in the context of chromatin. We will therefore also investigate how PARP1 DNA damage recognition, and in particular the structural dynamics are altered in the presence of nucleosomes. In summary using well defined smFRET experiments we will measure the structural dynamics during important steps of DNA damage recognition by PARP1. Our data will help to better understand functional differences of different PARP inhibitors and with that help the design of novel, more potent drugs.

• DFG Programme: Research Grants