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Expression, Purification, and Characterization of Putative Candida albicans Rad3, the Product of orf19.7119

Ki Moon Seong1,2#, Se Hyun Lee1#, Hag Dong Kim1, Chang Hoon Lee3, Hyesook Youn4, BuHyun Youn5*, and Joon Kim1*

1School of Life Sciences and Biotechnology and BioInstitute, Korea University, Seoul 136-713, Korea; E-mail: joonkim@korea.ac.kr

2Present address: Radiation Health Research Institute, Korea Hydro and Nuclear Power Co., Ltd., Seoul 132-703, Korea

3Department of Polymer Science and Engineering, Chosun University, Gwangju 501-759, Korea

4Department of Bioscience and Biotechnology/Institute of Bioscience, Sejong University, Seoul, 143-747, Korea

5College of Natural Sciences, Department of Biological Sciences, Pusan National University, Pusan 609-735, Korea; E-mail: bhyoun72@pusan.ac.kr

# These authors contributed equally to this work.

* To whom correspondence should be addressed.

Received October 4, 2010; Revision received December 30, 2010
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.
KEY WORDS: Candida albicans, Rad3, DNA repair, Fe-S cluster, functional complementation

DOI: 10.1134/S0006297911060071