생명과학회지 (Journal of Life Science)

  • 제14권2호
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  • Pages.325-330
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  • 2004
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
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진핵세포에서 DNA 회복에 관련된 HRD3 유전자의 분리, 발현 및 특성 연구

Study on Expression and Characterization of HRD3 Gene Related DNA Repair from Eukaryotic Cells

  • 발행 : 2004.04.01
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초록

효모에 있어 자외선에 의한 절제회복 관여 DNA회복유전자가 많이 알려져 있으나, 이들이 어떤 기능을 하는지는 아직 잘 알려져 있지 않다. 본 연구에서는 자외선 조사 시 절제회복의 초기 단계에 절대적으로 필요한 RAD3 유전자와 유사한 유전자인 HRD3 유전자를 분열형 효모인 Schizosaccharomyces pombe에서 분리하여 그 특성을 연구하였다. 이 결과 분리한 유전자는 효모 RAD3 유전자와 염기서열에서 약 70%이상의 유사성을 보였다. 이 유전자의 염기서열 결과 유전자 산물의 분자량은 75 kDa였다. 2-D gel 결과 과잉발현 시 HRD3 단백질은 숙주 단백질의 합성 억제 또는 분해 촉진을 유발하여 숙주세포인 대장균에 독성초과를 나타내었다. HRD3 유전자와 lacZ 유전자를 융합시킨 여러 가지 재조합 vector를 만들어 이들 융합단백질을 분리, 연구 한 결과 HRD3 단백질의 카르복실 말단부분이 효모에 있어서 DNA회복기능과 대장균에서의 독성효과를 나타내는 중요부위임이 확인되었다.

The RAD3 gene of Saccharomyces cerevisiae is required for excision repair and is essential for cell viability. RAD3 encoded protein possesses a single stranded DNA-dependent ATPase and DNA and DNA-RNA helicase activities. To examine the extent of conservation of structure and function of RAD3 during eukaryotic evolution, the RAD3 homolog gene was isolated by screening of genomic DNA library. The isolated gene was designated as HRD3 (Homologue of RAD3 gene). The over-expressed HRD3 protein was estimated to be a 75 kDa in size which is in good agreement with the estimated by the nucleotide sequence of the cloned gene. Two-dimensional gel electrophoresis showed that a number of other protein spots dramatically disappeared when the HRD3 protein was overexpressed. The overexpressed RAD3 protein showed a toxicity in E. coli host, suggesting that this protein may be involved in the inhibition of protein synthesis and/or degradation of host protein. To determine which part of HRD3 gene contributes to the toxicity in E. coli, various fusion plasmids containing a partial sequence of HRD3 and lac'Z gene were constructed. These results suggest that the C-terminal domain of HRD3 protein may be important for both toxic effect in E. coli and for its role in DNA repair in S. pombe.

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