Q1.Based on the information provided in Iyer et al. (2011) (you can also look up other sources), what reaction do deaminases catalyse? (1 mark). What is cytosine converted to when it is deaminated? (1 mark). Why might this conversion be toxic to a cell? (2 marks) (4 marks total)
In a recent paper (Mok et al., 2020), the authors describe the development of a method for base-editing of mitochondrial DNA (mtDNA), based on the enzymic action of DddA, the name they have given to the protein that contains pfam14428 (SCP1.201-like deaminase) in Burkholderia cenocepacia H111.
Q2.Based on the information provided in Mok et al., 2020, why is it important to develop a system that can provide precise targeted editing of mtDNA? (2 marks)
Q3.DddA catalyses the deamination of cytidines within dsDNA. What type of base conversion occurs as a result of this reaction (is it a transition or transversion)? (1 mark). Explain what this is. (1 mark).(2 marks total)
Mok and colleagues used DddA to create a CRISPR-free, RNA-free base editor that can install targeted mutations in the human mitochondrial genome. To do this they needed to determine which region of DddA conferred toxicity. In the paper, this region was then referred to as DddAtox.
Q4.Which amino acid region was identified as DddAtox. ? (Give the amino acid numbers for this region).(1 mark)
Mok et al. (20230) wanted to use DddAtox as a gene-editing tool, but the active protein (fused to programmable DNA-binding proteins) was toxic to the humancell lines they were using to determine whether DddAtox could edit the human mitochondrial genome.
Q5. What strategy did they adopt in order to use DddAtox to create a gene-editing tool without killing the humancell line cells?(1 marks)
As part of creating their gene-editing tool, Mok et al. included a uracil glycosylase inhibitor (UGI) in their construct.
Q6 What is the function of uracil DNA glycosylase (use databases to find this answer)? Why would Mok et al need to include a uracil glycosylase inhibitor in their gene-editing tool?(2 marks)
Q7. What is the significance of creating a CRISPR-free, RNA-free base editor, and what are its potential applications? Briefly explain, with reference to the ability to edit genes within mitochondrial DNA without requiring double-strand breaks. (5 marks)