Research
DNA-PROTEIN CROSSLINKS
Our research is focused on solving the mechanisms of DNA-protein crosslink repair in vitro, in cell culture and on the organismal level. We use CRISPR/Cas9 gene editing for gene Knock-Out as well as mutation introduction (Knock-In) in cell culture and zebrafish. Recently, we have expanded our focus on solving the DPC structures using the Cryo-EM.
DNA-protein crosslinks
DNA-protein crosslinks (DPCs) are a common type of DNA damage which appear when a protein forms an irreversible covalent bond with the DNA. Under physiological conditions these lesions are caused by reactive oxygen and nitrogen species, DNA helical alterations and increased aldehyde concentration due to histone demethylation, AlkB-type repair, amino acid metabolism, and lipid peroxidation. DPCs are also induced by exogenous sources such as UV light, ionizing radiation and chemotherapeutics. These lesions are very diverse because any protein in proximity to DNA can be crosslinked upon exposure to endogenous or exogenous crosslinking sources. DPCs are also intricately complex due to the diversity of crosslinking chemistries and protein sizes.
DPCs present a physical blockage to all DNA transactions: replication, transcription, recombination and repair and therefore the consequences of impaired DNA-Protein Crosslink Repair (DPCR) are severe. Considering their frequent occurrence and detrimental effect on all DNA transactions, it is not surprising that DPCs are implicated in aging, cardiovascular diseases, neurodegeneration and cancer. On a cellular level, aberrant DPC repair leads to the formation of DSBs, genomic instability and/or cell death, while on the organismal level impaired DPCR was so far shown to cause premature aging phenotypes and cancer.
Proteolysis-dependent DNA-Protein Crosslink Repair
The mechanism of DNA-Protein Crosslink Repair (DPCR) is still largely unknown. The reason wasn partly due to the misconception in the field that DPCs are repaired by canonical DNA damage repair pathways, nucleotide excision repair (NER) and homologous recombination (HR). However, we and others have recently shown that DPC repair is a specialised DNA damage repair pathway which relies on the proteolytic digestion of crosslinked proteins. The central players in the pathway are the metalloproteases Wss1 in yeast and SPRTN (or DVC1) in mammals which initiate DPCR by the proteolytic cleavage of crosslinked protein, followed by the removal of the protein remnant from DNA backbone via different downstream factors.
Zebrafish as a research model
Zebrafish is a well-established vertebrate model in developmental biology, toxicology and biomedicine. It offers a uniquely elegant toolbox to investigate the orchestration of DPC repair in vivo due to the ease of genetic manipulation and very fast developmental cycle. Additional advantages of the model include easy and automated fish maintenance, established methods for forward and reverse genetic studies and high quality of sequenced genome. Indeed, zebrafish is the only vertebrate whose genome is sequenced with similar quality to those of human and mouse. Given the high degree of evolutionary conservation between zebrafish and mammals, especially in respect to DNA damage related genes, utilizing the zebrafish model to understand the mechanisms of DNA repair pathways is an emerging field with proven benefits.
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(Image credit: MPI f. Developmental Biology/ P. Malhawar)
CRISPR/Cas9 genome editing
CRISPR/Cas9 genome editing is an efficient tool that revolutionized biological research. In zebrafish, CRISPR/Cas9-mediated gene manipulation is the method of choice for targeted mutagenesis as it surpasses the other two previously available methods, ZFNs and TALENs, in specificity and efficiency of germ line mutations. This year’s Nobel prize in Chemistry (2020) was awarded to Emmanuelle Charpentier and Jennifer Doudna for the discovery of ‘genetic scissors’. You can listen to the radio podcast at Croatian radio where dr. Popovic comments on this year’s Nobel prize at: https://radio.hrt.hr/aod/drag-mi-je-platon/356904/.
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#/)
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