Expression, purification, and characterization of putative Candida albicans Rad3, the product of orf19.7119

Ki Moon Seong, Se Hyun Lee, Hag Dong Kim, Chang Hoon Lee, Hyesook Youn, Buhyun Youn, Joon Kim

Research output: Contribution to journalArticle

2 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)666-676
Number of pages11
JournalBiochemistry (Moscow)
Volume76
Issue number6
DOIs
Publication statusPublished - 2011 Jun

Keywords

  • Candida albicans
  • DNA repair
  • Fe-S cluster
  • Rad3
  • functional complementation

ASJC Scopus subject areas

  • Biochemistry

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