Research Projects
Structural characterization of factors involved in DNA-protein crosslink repair
The project research goal is to elucidate the molecular mechanisms behind the DNA-protein crosslink repair pathway. 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. Obtained knowledge will be fundamentally important for a deeper understanding of the DPCR 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, our studies will offer novel approaches for investigating DNA repair complexes and disordered protein regions using cryo-EM.
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This project is a bilateral project jointly funded by Slovenian and Croatian Science Foundations (project number IPS-2020-01-4225, 2020-2023). A collaborative project includes two research groups: Marjetka Podobnik’s lab at the National Chemistry Institute in Ljubljana (Slovenia), and Popovic lab at Institute Ruder Boskovic in Zagreb, Croatia.
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Research Projects
The repair of topoisomerase DNA-protein crosslinks in vivo using zebrafish model
DNA-protein crosslinks (DPCs) are frequent DNA lesions caused by irreversible covalent bonds between proteins and DNA. These bulky lesions hinder all DNA transactions, and therefore the consequences of impaired DNA-Protein Crosslink Repair (DPCR) are severe. At the cellular level, defective repair causes DNA breaks, genomic instability, and cell death, while at the organismal level, impaired DPCR is associated with cancer, aging, and neurodegeneration. Topoisomerases (TOPs) are among the most abundant DPCs in cells under physiological conditions and TOP-DPC inducers such as topotecan and etoposide are used to treat various cancers. Tyrosyl-DNA phosphodiesterases (TDPs) are enzymes that remove crosslinked topoisomerase peptide residues from the DNA and their deficiency leads to neurological defects in mice and humans. Despite many efforts, the repair of these highly toxic DNA lesions is still not fully understood. We will investigate (1) which proteins are crucial for the repair of TOP1- and TOP2-DPCs in vivo and whether they are epistatic, (2) link between TOP-DPCR and the neurological defects in TDP-deficient organism, and (3) whether TOP-DPCs accumulate in the aging organism. The project is the first to investigate TOP-DPC repair at the organismal level. It will improve our understanding of human disease and aging and could lead to the identification of new drug targets for the treatment of cancer and neurological diseases.
Deciphering DNA-Protein Crosslink Repair in vivo using CRISPR/Cas9 genome editing in zebrafish model
To date, most aspects of DPCR are poorly understood including identification of the repair complexes, relationship between different pathways, how is the pathway choice made and the mechanistic link between impaired DPCR and cancer, aging and neurodegenerative diseases. So far, existing approaches have not addressed the orchestration of the DPCR pathways on the organismal level. Therefore, how defective DPC repair translates into disease phenotypes remains largely unknown. Recently, in the group, we have implemented zebrafish mutant and gene knock-out methods using CRISPR/Cas and fluorescent reporter assay for rapid germ line transmission screens and established novel methods to measure DPC repair in vivo using the zebrafish model. This approach represents a first step toward understanding the requirements for distinct repair complexes in different cellular and tissue-specific backgrounds. Using a range of techniques across the fields of molecular and cell biology and in vivo zebrafish model, we aim to unravel the mechanisms of DPC repair and try to understand how DPCs drive processes of aging, cancerogenesis and neurodegeneration.ccc