title: Expression, purification, and characterization of putative Candida albicans Rad3, the product of orf19.7119.
abstract: Invasive infections of Candida albicans are life-threatening clinical conditions affecting immunosuppressed patients. To maintain genome integrity and diversity, C. albicans utilizes DNA repair systems, such as nucleotide excision repair (NER), to escape from attack by macrophages. Rad3 helicase is a component of the TFIIH complex, which plays a role in transcription and the NER pathway. Accumulated evidence of studies from Archaea to humans has revealed that the conserved structure, including an iron-containing domain, is essential in the function of Rad3 helicase activity. However, no study of the Rad3 protein of C. albicans has yet been reported. In the present study, putative C. albicans Rad3 (CaRad3) has been cloned with orf19.7119 of the Candida genome. CaRad3 proteins were over-expressed and purified from E. coli and S. cerevisiae using a Ni-NTA column and a size exclusion column for physicochemical and functional characterization. Through EMR and spectrometric analysis, we have proven that the purified CaRad3 protein has a Fe-S cluster. We also revealed that CaRad3 protein has a helicase activity on a duplex DNA substrate. Furthermore, we showed that the CaRad3 protein purified from yeasts was N-glycosylated, and that this protein complemented the defects in both the NER pathway and transcription. These data suggest that the Rad3 helicase in C. albicans is the product of the orf19.7119 gene.
authors.affiliation: School of Life Sciences and Biotechnology and BioInstitute, Korea University, Seoul 136-713, Korea.
authors.forename: Ki Moon
authors.initials: KM
authors.lastname: Seong
authors.forename: Se Hyun
authors.initials: SH
authors.lastname: Lee
authors.forename: Hag Dong
authors.initials: HD
authors.lastname: Kim
authors.forename: Chang Hoon
authors.initials: CH
authors.lastname: Lee
authors.forename: Hyesook
authors.initials: H
authors.lastname: Youn
authors.forename: BuHyun
authors.initials: B
authors.lastname: Youn
authors.forename: Joon
authors.initials: J
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D002176:Candida albicans / Q000201:enzymology*; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D004247:DNA; D004265:DNA Helicases