Investigating Crosstalk Between DNA Repair and Ribosome Biogenesis
AbstractAlthough typically perceived as separate pathways, DNA repair and ribosome biogenesis have surprisingly been associated with one another by the reported interaction between the human DNA repair enzyme SMUG1 and the pseudouridine synthase dyskerin (DCK1, Cbf5 in yeast). Specifically, this interaction has been shown to excise 5-hydeoxylmethyl deoxyuridine, but not uridine or pseudouridine from RNA molecules.
Uracil glycosylases, such as SMUG1, remove damaged bases from DNA, leaving an abasic site open for repair. In particular, uracil glycosylases serve a role in protecting the genome from mutation in the event of cytosine deamination to uracil. Unrepaired U:G mismatches can give rise to transition mutations in replication, resulting in altered gene expression.
Dyskerin, a ribosome biogenesis factor, is part of a ribonucleoprotein complex that modifies ribosomal RNA by catalyzing the isomerization of uridine to pseudouridine. The function of dyskerin offers an interesting link to cancer, in which large ribosome quantities are generated within an enlarged nucleolus for rapid protein synthesis. Additionally, RNA pseudouridine synthases have been found to be overexpressed in many cancers and linked to a poor prognosis. Following the overexpression and purification of SMUG1 and Cbf5 (dyskerin homologue), the study looks to establish an electrophoresis and fluorescence based uracil-DNA glycosylase assay utilizing fluorescently labelled DNA molecules and DNA endonucleases. In light of the unclear SMUG1-dyskerin interaction, this fluorescence based assay, will be used to characterize the effect of Cbf5 on SMUG1 activity and vice versa. The affinity of SMUG1 to RNA in the presence and absence of Cbf5 will also be investigated via binding studies, with the aim of testing the hypothesis that dyskerin acts as an RNA tether for SMUG1.
Studying these protein-protein and RNA-protein interactions will provide insight into the crosstalk between the two important, and previously thought to be unrelated, cellular pathways of DNA repair and ribosome biogenesis with potential implications for new cancer treatment strategies.