DNA-protein crosslinks (DPCs) are severe DNA lesions which occur when a protein becomes irreversibly covalently linked to DNA. They have adverse effects on the organismal level including cancer, premature aging and neurodegenerative diseases. Due to their bulky nature, DPCs impair all DNA transactions (replication, transcription and repair) and DPC repair is therefore an essential cellular pathway. Despite the fact that DPCs are frequently occurring in the nucleus and cause severe damage on a cellular level, not much is known about their repair mechanisms at a molecular level. Our structural studies aim to solve near-atomic details of the SPRTN-dependent DPC repair complex, the SPRTN:p97 complex and a novel DPC factor, ACRC, for the first time. Obtained knowledge will be fundamentally important for a deeper understanding of the repair pathway and thus for the developing research on p97 and SPRTN inhibitors for the purpose of targeted clinical therapies in cancerogenesis and aging. In terms of methodology, proposed studies will offer novel approaches for investigating DNA repair complexes and disordered protein regions using cryo-EM. Cryo-EM (Cryogenic electron microscopy) is a powerful approach to solve structures of large proteins and protein complexes. The importance of the approach was further emphasized in 2017., when the Nobel Prize in Chemistry was awarded to Jacques Dubochet, Joachim Frank, and Richard Henderson "for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution." (https://www.nature.com/news/cryo-electron-microscopy-wins-chemistry-nobel-1.22738;https://www.chemistryworld.com/news/explainer-what-is-cryo-electron-microscopy/3008091.article#/)

DNA-protein crosslink (DPC) is a type of DNA lesion where a protein becomes irreversibly covalently bound to DNA upon exposure to endogenous or exogenous crosslink inducers. Endogenous DPC inducers are products of normal cellular metabolism such as reactive oxygen species, aldehydes and DNA helical alterations, while exogenous inducers include UV light, ionizing radiation and various chemicals. DNA-protein crosslinks are common DNA lesions which present a physical blockage to all DNA transactions: replication, transcription, recombination and repair. If not repaired, DPCs cause genomic instability and adverse phenotypes in humans including premature aging, neurodegeneration and cancer. Despite the frequency and severe outcomes of DPCs, DNA-protein crosslink repair (DPCR) has been sparsely studied, mostly because it has not been considered a separate DNA damage repair pathway until recently. In 2014 and 2016, several groups have identified a novel proteases, Wss1 and SPRTN, which initiate the removal of DPCs through the proteolytic digestion of crosslinked proteins. The discovery of proteolysis-coupled DPC repair lead to recognition of the DNA-protein crosslink repair as a separate DNA damage repair pathway. However, we currently do not know how is the pathway orchestrated and which other factors are involved, while almost nothing is known of DPCR mechanism in vivo. Therefore, within this project we aim to unravel the orchestration of the DPCR pathway in vivo using zebrafish (Danio rerio) as a well-characterized vertebrate model. We will use CRISPR/Cas9 gene manipulation tools to knock-out or mutate specific genes in zebrafish which we suspect are involved in the removal of DNA-protein crosslinks. Contribution of each protein (and their combinations) to the DNA-protein crosslink repair will be quantified after DPC isolation from transgenic zebrafish embryos and adults.

Identification and functional characterization of (eco)toxicologically relevant polyspecific membrane transport proteins in zebrafish (Danio rerio)

                                                                                                                                                                                                                                                                                                                             A coordinated system of transport proteins, channels, receptors and enzymes act as cellular gatekeepers to foreign molecules, critically determining the so-called ADME-Tox (Absorption, Distribution, Metabolism, Excretion – toxicity) properties of a molecule. The polyspecific uptake and efflux transmembrane proteins are essential components of this complex cellular defense and detoxification/xenobiotic processing machinery in mammals, highly important and widely recognized in the context of pharmacology and human toxicology. However, they are scarcely investigated in non-mammalian species, and are not adequately addressed in the field of environmental toxicology. Consequently, the major goal of the proposed project is identification and detailed functional characterization of novel, (eco)toxicologically relevant uptake and efflux transport proteins that are not addressed so far in the context of environmental toxicology nor in non-mammalian species in general. Our research will be focused on selected polyspecific uptake transport proteins from the SLC21 and SLC22 (Solute Carriers) families, efflux transporters from the MATE (Multidrug and Toxic Extrusion) family, and finally, on the RLIP76 as the most recently discovered stress-responsive, multi-functional membrane protein. We will use zebrafish (Danio rerio) as a highly relevant vertebrate research model. Our methodological approach will be based on several subsequent research phases: phylogenetic and gene expression analyses; transfection studies in appropriate heterologous expression system(s); transport-activity assays; analyses of the transport mechanism and structural properties; high-throughput-screening for the identification of interactors of selected transporters among environmental contaminants; and finally, in vivo evaluation of the (eco)toxicological relevance of selected transporters using the zebrafish functional genomics tools.

http://www.irb.hr/nato-savariver/

Croatia (Ministry of Science Education and Sports) – Germany (DAAD) bilateral project

EC FP6 Project

EC FP6 Project

Project supported by the Croatian Ministry of Science and Technology, project No P0098135

Project supported by the Government of the Republic of Croatia

Project supported by the Croatian Ministry of Science Education and Sports

Marie Curie Initial Training Networks (ITN) project

Swiss National Science Fondation (SNSF), SCOPES Joint Research Project

http://www.snf.ch/E/international/europe/scopes/Pages/default.aspx