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Functional and mechanistic investigations into the importance of PADI enzymes for DNA repair

Principal Supervisor: Dr Clare Davies

Secondary Supervisor(s): Prof Jo Morris

University of Registration: University of Birmingham

BBSRC Research Themes:

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Deadline: 4 January, 2024


Project Outline

The ability for a cell to repair damaged DNA is incredibly important for maintaining genome stability. An inability to do so leads to mutagenic events that can predispose individuals to neurological disease and cancer. Whilst our knowledge into the key proteins plays in various DNA repair pathways has increased dramatically, it is becoming apparent that protein post-translational modifications play an integral role. Current thinking is that these modifications act as a platform, promoting protein complex assembly and disassembly, modulating enzymatic activity, and signaling pathway choice.

The amino acid arginine is targeted for methylation by the PRMT family of enzymes, and the Davies lab has recently described a role for PRMT5 in the DNA damage response (Clarke et al., Molecular Cell, 2017). Interestingly, arginine is also targeted by a second family of enzymes called PADIs (peptidylarginine demininases). Here, PADIs post-translationally convert positively charged arginine in substrate proteins to the neutral nonessential amino acid citrulline, a process referred to as citrullination. Surprisingly, very little is known about PADI enzymes for DNA repair even though PADI2 and PADI4 levels are elevated in cancer possibly leading to chemoresistance. Top-down mass spectrometry screens for the protein citrullinome has identified an enrichment of DNA repair proteins. Supporting this, we now have data that PADI enzymes are indeed important for maintain genome stability. However, the mechanisms by which this occurs, the specific type of DNA repair pathway that required PADI activity, or the substrates they citrullinate are still unknown.

In this PhD studentship, we will address the following important questions:

  1. Are PADI2/PADI4 driving a specific DNA repair pathway? Achieved thought specific PADI2 and PADI4 inhibitors, in parallel with CRISPR-mediated PADI2/PADI4 knockout and overexpression studies after ionising radiation (double strand breaks), MMC (interstrand crosslinks/FA pathway), HU/Aphidicolin (replication stress) and campothecin (Top1 cleavage complex). Methodologies include colony survival assays, DNA damage induction as measure by gH2AX and 53BP1 foci, DNA fibre analysis, signalling pathway activation (western blotting).
  2. Which DNA repair proteins are citrullinated after DNA damage? Using probes that specifically detect citrullinated proteins, we will conduct Mass Spectrometry after DNA damage in wildtype or PADI2 or PAD4 knockout cells.
  3. How does how citrullination of a DNA repair protein affects its function? Taking proteins identified from question 2, we will map the citrullination sites (by mass spec) and conduct in silico structural analysis to understand how arginine-citrulline conversion affects structure. Genetic engineering of mammalian cells to express a photo-activated site-specific incorporation of citrulline will establish in vivo relevance.

Given that PADI inhibitors are currently being explored for the treatment of inflammatory disease, understanding how PADIs contribute to DNA repair and genome stability could lead to drug repurposing in combination with conventional DNA damaging chemotherapies, in PADI2/4 overexpressing cancers

References

1) Protein Arginine Deiminases (PADs): Biochemistry and Chemical Biology of Protein Citrullination. Mondal S, Thompson PR.Acc Chem Res. 2019 Mar 19;52(3):818-832. doi: 10.1021/acs.accounts.9b00024. Epub 2019 Mar 7.PMID: 30844238

2) Insights into peptidylarginine deiminase expression and citrullination pathways.Yu K, Proost P.Trends Cell Biol. 2022 Sep;32(9):746-761. doi: 10.1016/j.tcb.2022.01.014. Epub 2022 Feb 21.PMID: 35197210

Techniques

  • Cell culture, siRNA knockdown, drug treatments
  • Molecular cloning including site-directed mutagenesis
  • CRISPR-Cas9-mediated gene editing
  • Lentiviral infections to generate cell lines
  • Colony survival assays
  • Immunofluorescene of DNA damage foci
  • DNA fibre analysis
  • Western blotting
  • Immunoprecipitations
  • Mass Spectrometry and associated analysis
  • In silico structural analysis
  • Photoactivatable citrulline incorporation in mammalian cells
  • In silico structure analysis