Projects / Programmes
Structural characterization of factors involved in DNA-protein crosslink repair
| Code |
Science |
Field |
Subfield |
| 1.05.00 |
Natural sciences and mathematics |
Biochemistry and molecular biology |
|
| Code |
Science |
Field |
| 1.06 |
Natural Sciences |
Biological sciences |
DNA damage, DNA-protein crosslinks (DPCs), DPC repair mechanisms, cancer, ageing, SPRTN protease, p97 AAA ATPase, ACRC/GCNA protein, cryo-electron microscopy, x-ray crystallography, structural studies
Researchers (10)
| no. |
Code |
Name and surname |
Research area |
Role |
Period |
No. of publicationsNo. of publications |
| 1. |
55098 |
Maksimiljan Adamek |
Biochemistry and molecular biology |
Technical associate |
2021 - 2022 |
18 |
| 2. |
55978 |
Primož Bembič |
Biochemistry and molecular biology |
Technical associate |
2023 - 2024 |
5 |
| 3. |
31814 |
Urška Dečko |
|
Technical associate |
2020 - 2021 |
3 |
| 4. |
50722 |
Luka Kavčič |
|
Technical associate |
2022 - 2024 |
29 |
| 5. |
38255 |
PhD Andreja Kežar |
Biochemistry and molecular biology |
Researcher |
2020 - 2024 |
42 |
| 6. |
35382 |
PhD Matic Kisovec |
Biochemistry and molecular biology |
Researcher |
2020 - 2024 |
80 |
| 7. |
55663 |
Tanja Peric |
|
Technical associate |
2021 - 2024 |
4 |
| 8. |
12048 |
PhD Marjetka Podobnik |
Biochemistry and molecular biology |
Head |
2020 - 2024 |
317 |
| 9. |
38473 |
Tomaž Švigelj |
|
Technical associate |
2020 - 2022 |
11 |
| 10. |
21684 |
Tea Tomšič |
|
Technical associate |
2020 |
2 |
Organisations (1)
| no. |
Code |
Research organisation |
City |
Registration number |
No. of publicationsNo. of publications |
| 1. |
0104 |
National Institute of Chemistry |
Ljubljana |
5051592000 |
20,997 |
Abstract
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. DPCs 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 novel proteases, Wss1 in yeast and SPRTN in mammals, which initiate the removal of DPCs through proteolytic digestion of crosslinked proteins. The discovery of proteolysis-coupled DNA repair lead to recognition of the DPCR as a separate DNA damage repair pathway. However, molecular mechanisms and structural knowledge behind the protease-mediated DPCR is lacking. To date structural information on DPCs and DPCR factors is limited to a fragment containing the active site of SPRTN. Data from yeast indicate that SPRTN might work in concert with the ATP-dependent AAA family segregase p97, another essential protein linked to DPCR. In addition, we and others have discovered another putative protease, ACRC (syn. GCNA) harboring a catalytic domain similar to SPRTN. Currently, structural data on SPRTN interaction with DPC substrates and p97 segregase is lacking, as well as biochemical and structural data on ACRC, a potential new protease involved in DPCR. We aim to (1) characterize the SPRTN:p97 complex and ACRC in vitro and in vivo, and (2) solve three-dimensional structures of human SPRTN:DPC and SPRTN:p97 complexes as well as of the ACRC protein, using cryo-electron microscopy (cryo-EM) and X-ray crystallography, respectively. SPRTN complexes with a model DPC and p97 will be reconstituted in vitro. To generate a homogenous population of DPCs we will use Ogg1-DNA adducts that can be generated in vitro at high efficiency. A state-of-the-art cryo-EM facility harboring a cryo-electron microscope with phase plate and a direct electron detector will be used to obtain high-resolution structures, even for complexes with higher degree of flexibility, while X-ray crystallography will be used to solve ACRC structure due to the small size of the protein. The proposed research will unravel the mechanisms underlying DPCR on a molecular level. We envision that obtained knowledge will be fundamentally relevant, especially in the context of emerging importance of p97 and SPRTN inhibitors in cancer treatment and aging. The project will be performed by closely connected research teams, one from Institute Ruđer Bošković, Croatia, and the other from the National Institute of Chemistry, Slovenia. The teams share common research interest in deciphering molecular interactions and mechanisms of biological processes, including knowledge in proteolysis, protein-protein and protein-nucleic acid interactions. These common research interests and expertise will be of a great importance for the success of this project. Moreover, complementarity between the teams in terms of methodological approaches is and additional critical factor that will importantly add to the success of this proposal, as well as the transfer of knowledge between the collaborating international teams.
