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 language | English |
|---|---|
| Pages (from-to) | 666-676 |
| Number of pages | 11 |
| Journal | Biochemistry (Moscow) |
| Volume | 76 |
| Issue number | 6 |
| DOIs | |
| Publication status | Published - 2011 Jun 1 |
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Keywords
- Candida albicans
- DNA repair
- Fe-S cluster
- functional complementation
- Rad3
ASJC Scopus subject areas
- Biochemistry
Cite this
Expression, purification, and characterization of putative Candida albicans Rad3, the product of orf19.7119. / Seong, Ki Moon; Lee, Se Hyun; Kim, Hag Dong; Lee, Chang Hoon; Youn, Hyesook; Youn, Buhyun; Kim, Joon.
In: Biochemistry (Moscow), Vol. 76, No. 6, 01.06.2011, p. 666-676.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Expression, purification, and characterization of putative Candida albicans Rad3, the product of orf19.7119
AU - Seong, Ki Moon
AU - Lee, Se Hyun
AU - Kim, Hag Dong
AU - Lee, Chang Hoon
AU - Youn, Hyesook
AU - Youn, Buhyun
AU - Kim, Joon
PY - 2011/6/1
Y1 - 2011/6/1
N2 - 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.
AB - 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.
KW - Candida albicans
KW - DNA repair
KW - Fe-S cluster
KW - functional complementation
KW - Rad3
UR - http://www.scopus.com/inward/record.url?scp=79959734111&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=79959734111&partnerID=8YFLogxK
U2 - 10.1134/S0006297911060071
DO - 10.1134/S0006297911060071
M3 - Article
C2 - 21639847
AN - SCOPUS:79959734111
VL - 76
SP - 666
EP - 676
JO - Biochemistry. Biokhimiia
JF - Biochemistry. Biokhimiia
SN - 0006-2979
IS - 6
ER -