The role of Spartan in DNA replication - SprtnRep
(HRZZ Research project IP-06-2016)
: 01.09.2017.- 31.08.2021.
Kristijan Ramadan, Oxford University, Institute of oncology, Oxford, UK
Janoš Terzić, University od Split, School of Medicine, Split, Croatia
Jasminka Omerović, University od Split, School of Medicine, Split, Croatia
Matilda Šprung, University od Split, Faculty of science, Split, Croatia
mag. biol. mol. Anja Ančić
The maintenance of chromosomal stability during DNA replication and repair is crucial for preventing cell transformation from normal to pathogenic state; defects in DNA repair mechanisms cause developmental abnormalities, premature aging, and cancer. Most types of DNA damage block the progression of replication forks, because DNA polymerases cannot recognize damaged nucleotides. At sites of stalled DNA replication fork, replication sliding clamp protein PCNA becomes ubiquitinated and serves as a molecular marker for initiating translesion DNA synthesis (TLS). Recently, my colleagues and I have identified germline mutations in SPRTN gene (Spartan) in three patients, who developed progeroid syndrome characterized by genomic instability and early onset hepatocellular carcinoma (HCC). Previously it was proposed that Spartan is important for activation of TLS (Ghosal et al, 2012; Kim et al, 2013). However, our results show that Spartan plays an essential role in normal DNA replication by preventing DNA replication-stress and maintaining genome stability (Marinovic-Terzic et al, 2014). Here we propose that Spartan is a metaloprotease important for removal of DNA-protein crosslinks, thus enabling the progression of replication forks.
Our goal in this project is to:
1. Define the functions of Spartan’s domains,
2. Determine Spartans interacting properties/partners, and
3. Describe the dynamics of a DNA damage repair process in Spartan depleted cells.
Most of the techniques we will use in this work are established procedures in our laboratories: Western blot, IP, GST-pull down, immunofluorescent microscopy, RNAi, SILAC based mas spectrometry and flow cytometry. One novel technique will be used as well – CRISPR/Cas9 targeted gene editing. All experiments will be performed using cultured cells. This project proposal has a potential to generate very interesting results with high possibility for publications in top scientific journals. Results of this project will help to better understand the specific role of Spartan in DNA replication and genome stability.