Biochemical and cellular characterization of DDX59 helicase that is associated oral-facial-digital syndrome
Date
2020-10-01
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
Type
Thesis
Degree Level
Masters
Abstract
DDX59 belongs to the DEAD-box helicase subfamily, and mutations in DDX59 are associated with Oral-facial-digital syndrome (OFDS) that is characterized by malformations of the face, oral cavity, and digits. DDX59 is one of the sixteen genes that have been reported to be involved in various subtypes of OFDS. Most OFDS-associated proteins are localized primarily to cilia components, and influencing ciliogenesis and ciliary functions. However, the effect of DDX59 mutations in OFDS and their links to ciliogenesis remained unidentified. In this study, we overexpressed the human DDX59 gene in bacteria and purified the protein using nickel affinity and size exclusion chromatography. The DDX59 protein identity was confirmed by Western blotting analysis using a DDX59 protein specific-antibody. Although the protein sequence and domain organizations suggest DDX59 is an RNA helicase, surprisingly, our in vitro helicase assays revealed that DDX59 was ineffective on the RNA substrates tested. In contrast, DDX59 exhibited ATP dependent 3′→5′ DNA helicase activity. Interestingly, the OFDS-associated DDX59 mutations, V367G and G534R, reduced the DNA helicase activity, indicating that the loss-of-function might be the underlying cause of OFDS. Using the CRISPR/Cas9 system, an attempt to generate DDX59 knockout in the human retinal pigmented epithelial (RPE1) cell line resulted in heterozygous knockouts, but not the homozygous knockouts, indicating DDX59 is essential for the cell survival. In spite of retaining only one wild-type allele, our immunofluorescence analysis showed a remarkable decrease in the number of ciliated cells upon DDX59 depletion, suggesting that DDX59 is necessary for the formation of primary cilia. Taken together, we found that DDX59 is an ATP dependent 3′→5′ DNA helicase and an ineffective RNA helicase, and that its loss-of-function might lead to the pathogenesis of OFDS through defective ciliogenesis.
Description
Keywords
DEAD-box helicase, DDX59 protein, DNA helicase, RNA helicase, ATP hydrolysis, DDX59 mutations, CRISPR-Cas9 system, DDX59-knockout, Oral-facial-digital-syndrome, Ciliopathies, RPE1 cells, Primary cilia, ciliary formation
Citation
Degree
Master of Science (M.Sc.)
Department
Biochemistry
Program
Biochemistry