Nuclear actin-related proteins (ARPs) are stoichiometric subunits of chromatin remodelers and specifically recognize and bind to (modified) histone proteins. In the case of the chromatin modifiers INO80, SWR1 and NuA4, canonical monomeric actin is also an important constituent of these large macromolecular complexes and actively takes part in the remodeling reaction. The main characteristic of actin, however, is its dynamic nature and its capacity to form polymeric actin filaments, which is tightly regulated by a plethora of actin binding proteins amongst them also nuclear ARPs. Interestingly, alteration of actin levels strongly sensitize cells to DNA damage in an enigmatic process and the chromatin-binding nuclear ARPs are obviously strong candidates that possibly couple actin homeostasis in the nucleus with a putative role in genomic integrity. Missing from the field is a genetic approach to specifically mutate actin in the chromatin modifier context and to analyse the impact of polymerization mutants in genomic integrity. The central hypothesis in this research proposal is that nuclear ARP/chromatin remodeler associated actin is a key mediator in genomic maintenance. This assumption is based on the very recent finding in the Gasser laboratory that interference with actin homeostasis renders cells to be hypersensitive to genomic insult as chromosomes are rapidly fragmented in presence of actin depolymerizing agent Latrunculin A and DNA damaging agent zeocin. The major challenge in this project is to bypass the paramount role of actin in the cytoskeleton and to focus on the fraction of nuclear ARP associated actin.
This will be achieved by genetically replacing nuclear ARPs with ARP-actin fusion constructs in the budding yeast S. cerevisiae, which will allow me to introduce actin mutants exclusively in a chromatin modifying context. Concomitantly, a new approach to generate actin probes for microscopy based on bicyclic peptides will be applied.
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