DNA damage poses a serious threat to human health, as it may cause cancer and various other diseases. DNA repair proteins need to arrive at the site of DNA damage at the right time and in a precise manner, just like an ambulance arriving at the site of a car crash. To ensure this correct timing, multiple proteins need to interact with each other. Such interactions are oftentimes mediated via the addition of small molecules – called post-translational modifications - to a protein. One of the many post-translational modifications is poly(ADP-ribosylation) or PARylation, which is often found at DNA damage sites. PARylation regulates a great variety of cellular functions such as DNA replication and repair, transcription, apoptosis, development, differentiation and aging. The addition of PAR is catalyzed by the action of PAR polymerase (PARP), while its removal is carried out mostly by a different enzyme called PARG.
In their recent publication in Nucleic Acids Research, Dea Slade and her team at MFPL report the identification of the mechanism of interaction of PARG with an important protein in DNA repair – PCNA, a DNA clamp. “While much has been known about the mechanism of PARG enzymatic activity, our findings provide the first step towards understanding the mechanism and regulation of interactions of this essential mammalian enzyme. Fruitful collaborations with MFPL and Vienna Biocenter (VBC) groups (Djinovic-Carugo, Zagrovic, Hartl, Gotzmann, Elsayad, Mechtler) have been critical for the successful completion of this project. Our plan is to extend our findings to other PCNA-interacting proteins in order to understand how dynamic interactions with PCNA are regulated by post-translational modifications”, says Dr. Slade.
Dea Slade, together with Kareem Elsayad from VBCF Advanced Microscopy and Josef Gotzmann from MFPL BioOptics, recently won a WWTF Next grant, which will support her work on the DNA damage response with approximately €49.000 for one year.
As part of the WWTF Life Sciences 2014 – Imaging program, the tripartite team has developed a new imaging system tailored to their experimental needs: a controllable UV add-on module for a so-called “confocal time-resolved fluorescence microscope”. This experimental configuration allows them to induce DNA damage via UV laser and investigate and quantify the dynamics of protein interactions at a single cell level at the same time – a new technique, which they now hope to extend to a commercial setting thanks to their WWTF Next grant.
“As an important step towards validation of this new system, we will reach out to the scientific community to test known DNA damage-related protein-protein interactions, particularly those that may be relevant in the context of cancer therapy”, says Dea Slade.
Publication in Nucleic Acids Research:
Tanja Kaufmann, Irina Grishkovskaya, Anton A. Polyansky, Sebastian Kostrhon, Eva Kukolj, Karin M. Olek, Sebastien Herbert, Etienne Beltzung, Karl Mechtler, Thomas Peterbauer, Josef Gotzmann, Lijuan Zhang, Markus Hartl, Bojan Zagrovic, Kareem Elsayad, Kristina Djinovic-Carugo and Dea Slade: A novel non-canonical PIP-box mediates PARG interaction with PCNA. In: Nucleic Acids Research (July 2017). DOI: doi.org/10.1093/nar/gkx604