Biochemical characterization of C15ORF41: a novel nuclease

Thesis

Congenital dyserythropoietic anaemia type I (CDA-I) is a rare autosomal recessive form of anaemia that is characterized by ineffective erythropoiesis and abnormal morphologies of bone marrow erythroblasts. While previous studies have indicated mutations in the CDAN1 gene for a large group of patients with CDA-I, new research has revealed that mutations in the C15ORF41 gene are also linked to patients with the condition.

With the recent purification of the C15ORF41 protein by the Structural Genomics Consortium at the University of Oxford, a series of biochemical assays were conducted in order to characterize the catalytic and mechanistic behaviour of the protein. Nuclease assays indicated that the protein, when co-purified with the CDAN1 protein Codanin1, acts as a single-stranded endonuclease that acts on both DNA and RNA substrates. These assays also revealed that the complex incises in 5-6 nucleotide increments in a 3’-to-5’ directionality and exhibits a preference for 3’ flaps. Electrophoretic Mobility Shift Assays indicated that there is likely a 2:1 protein to substrate stoichiometry. Fluorescence anisotropy data showed that the C15ORF41-Codanin complex appeared to have much higher affinity to DNA substrates than the individual constituent proteins.

The results of this study provided initial evidence into the predicted endonucleolytic behaviour of the protein and established a solid foundation for further investigations of the mechanistic basis of its activity. This is absolutely critical for the molecular understanding of CDA-I for the formulation of future treatments, and is the first step in examining a novel endonuclease that could further our grasp on DNA damage repair proteins.

Authors: Ezzibdeh, R

• DOI: 10.5287/ora-zrgavwe5q
• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford