Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
권호 표지

Phenotypic Suppression of Rad53 Mutation by CYC8

CYC8에 의한 rad53 돌연변이의 표현형 억제에 대한 연구

  • Park, Kyoung-Jun (Department of Biological Sciences, Inha University) ;
  • Choi, Do-Hee (Department of Biological Sciences, Inha University) ;
  • Kwon, Sung-Hun (Department of Biological Sciences, Inha University) ;
  • Kim, Joon-Ho (Department of Biological Sciences, Inha University) ;
  • Bae, Sung-Ho (Department of Biological Sciences, Inha University)
  • 박경준 (인하대학교 자연과학대학 생명과학과) ;
  • 최도희 (인하대학교 자연과학대학 생명과학과) ;
  • 권성훈 (인하대학교 자연과학대학 생명과학과) ;
  • 김준호 (인하대학교 자연과학대학 생명과학과) ;
  • 배성호 (인하대학교 자연과학대학 생명과학과)
  • Received : 2009.02.26
  • Accepted : 2010.03.15
  • Published : 2010.06.30
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Abstract

RAD53 functions as an effector kinase of checkpoint pathways in Saccharomyces cerevisiae, which plays a central role to regulate many downstream cellular processes in response to DNA damage. It also involves in transcriptional activation of various genes including RNR genes which encode the key enzyme required for dNTP synthesis. In this study, we identified CYC8 as a suppressor for the hydroxyurea sensitivity of $rad53{\Delta}$ mutation. $Rad53{\Delta}$ mutant transformed with a multi-copy plasmid containing CYC8 showed increased hydroxyurea resistance. In contrast, TUP1 which forms a complex with CYC8 did not function as a suppressor. In the case of mutations, both $cyc8{\Delta}$ and $tup1{\Delta}$ suppressed hydroxyurea sensitivity of $rad53{\Delta}$. Since CYC8 can propagate as a prion in yeast, overexpression of CYC8 induced misfolding of the normal CYC8 proteins, resulting in dominant cyc8-phenotype. Therefore, it is suggested that CYC8 can act as a multi-copy suppressor due to its prion property. It was observed that the levels of RNR transcription were increased in the yeast strains containing either multi-copies of CYC8 gene or $cyc8{\Delta}$ mutation, suggesting that the increased level of RNR will elevate the intracellular pools of dNTPs, which, in turn, suppress the phenotype of $rad53{\Delta}$ mutation.

RAD53은 효모의 검문지점 경로가 DNA 손상을 감지하여 여러 가지 후속적인 세포 내 반응을 일으키는 데 핵심적인 역할을 하는 인산화 효소일 뿐만 아니라, dNTP 생성에 중요한 RNR 유전자 등의 전사 활성화 과정에도 관여하는 효모의 생존에 필수적인 유전자이다. 본 연구에서는 rad53${\Delta}$ 돌연변이의 hydroxyurea에 대한 민감성을 억제하는 억제자로서 CYC8을 동정하였다. CYC8 유전자가 많은 사본으로 존재할 때 rad53${\Delta}$ 균주의 hydroxyurea에 대한 내성이 증가하였으나, CYC8과 복합체로 작용하는 TUP1은 다사본 억제자로 작용하지 못하였다. 반면, 삭제 돌연변이의 경우, cyc8${\Delta}$과 tup1${\Delta}$ 모두 억제자로 작용하였다. CYC8은 효모에서 프리온 단백질로 작용하기 때문에 과량 발현되면 정상적인 CYC8 단백질의 잘못된 접힘을 유발하게 되고, 결과적으로 우성의 $cyc8^-$ 표현형이 나타나게 된다. 따라서 CYC8이 다사본 억제자로 작용하는 이유는 이러한 프리온의 특성 때문으로 추측된다. CYC8이 다사본이거나 cyc8${\Delta}$ 돌연변이일 경우 모두 RNR 유전자의 전사가 증가되는 것을 관찰하였다. 따라서 CYC8에 의한 rad53${\Delta}$ 돌연변이의 억제는 RNR 증가에 따른 세포 내 dNTP 증가 때문으로 생각된다.

Keywords

References

  1. Baudin, A., O. Ozier-Kalogeropoulos, A. Denouel, F. Lacroute, and C. Cullin. 1993. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 21, 2239-3330.
  2. Ben-Yehoyada, M., J. Gautier, and A. Dupre. 2007. The DNA damage response during an unperturbed S-phase. DNA Repair 6, 914-922. https://doi.org/10.1016/j.dnarep.2007.02.005
  3. Branzei, D. and M. Foiani. 2005. The DNA damage response during DNA replication. Curr. Opin. Cell. Biol. 17, 568-575. https://doi.org/10.1016/j.ceb.2005.09.003
  4. Branzei, D. and M. Foiani. 2006. The Rad53 signal transduction pathway: replication fork stabilization, DNA repair, and adaptation. Exp. Cell Res. 312, 2654-2659. https://doi.org/10.1016/j.yexcr.2006.06.012
  5. Chabes, A., B. Georgieva, V. Domkin, X. Zaho, R. Rothstein, and L. Thelander. 2003. Survival of DNA damage in yeast directly depends on increased dNTP levels allowed by relaxed feedback inhibition of ribonucleotide reductase. Cell 112, 391-401. https://doi.org/10.1016/S0092-8674(03)00075-8
  6. Chabes, A. and B. Stillman. 2007. Constitutively high dNTP concentration inhibits cell cycle progression and the DNA damage checkpoint in yeast Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 104, 1183-1188. https://doi.org/10.1073/pnas.0610585104
  7. Chen, S., M.B. Smolka, and H. Zhou. 2007. Mechanism of Dun1 activation by Rad53 phosphorylation in Saccharomyces cerevisiae. J. Biol. Chem. 282, 986-995. https://doi.org/10.1074/jbc.M609322200
  8. Choi, D.H., Y.M. Oh, S.H. Kwon, and S.H. Bae. 2008. The mutation of a novel Saccharomyces cerevisiae SRL4 gene rescues the lethality of rad53 and lcd1 mutations by modulating dNTP levels. J. Microbiol. 46, 75-80. https://doi.org/10.1007/s12275-008-0013-6
  9. Chris, K., S. Michaelis, and A. Mitchell. 1994. Methods in yeast genetics. pp. 207-217. In CSHL press
  10. Elledge, S.J. and R.W. Davis. 1990. Two genes differentially regulated in the cell cycle and by DNA-damaging agents encode alternate regulatory subunits of ribonucleotide reductase. Genes Dev. 4, 740-751. https://doi.org/10.1101/gad.4.5.740
  11. Elledge, S.J., Z. Zhou, J.B. Allen, and T.A. Navas. 1993. DNA damage and cell cycle regulation of ribonucleotide reductase. Bioessays 15, 333-339. https://doi.org/10.1002/bies.950150507
  12. Huang, M. and S.J. Elledge. 1997. Identification of RNR4, encoding a second essential small subunit of ribonucleotide reductase in Saccharomyces cerevisiae. Mol. Cell Biol. 17, 6105-6113. https://doi.org/10.1128/MCB.17.10.6105
  13. Huang, M., Z. Zhou, and S.J. Elledge. 1998. The DNA replication and damage checkpoint pathways induce transcription by inhibition of the Crt1 repressor. Cell 94, 595-605. https://doi.org/10.1016/S0092-8674(00)81601-3
  14. Koc, A. and G.F. Merrill. 2007. Checkpoint deficient rad53-11 yeast cannot accumulate dNTPs in response to DNA damage. Biochem. Biophys. Res. Commun. 353, 527-530. https://doi.org/10.1016/j.bbrc.2006.12.049
  15. Melo, J. and D. Toczyski. 2002. A unified view of the DNAdamage checkpoint. Curr. Opin. Cell Biol. 14, 237-245. https://doi.org/10.1016/S0955-0674(02)00312-5
  16. Patel, B.K., J. Gavin-Smyth, and S.W. Liebman. 2009. The yeast global transcriptional co-repressor protein Cyc8 can propagate as a prion. Nat. Cell Biol. 11, 344-349. https://doi.org/10.1038/ncb1843
  17. Sikorski, R. and P. Hieter. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19-27.
  18. Smith, R.L. and A.D. Johnson. 2000. Turning genes off by Ssn6- Tup1: a conserved system of transcriptional repression in eukaryotes. Trends Biochem. Sci. 25, 325-330. https://doi.org/10.1016/S0968-0004(00)01592-9
  19. Wang. P.J., A. Chabes, R. Casagrande, X.C. Tian, L. Thelander, and T.C. Huffaker. 1997. Rnr4p, a novel ribonucleotide reductase small-subunit protein. Mol. Cell Biol. 7, 6114-6121.
  20. Zhao, X., E.G.D. Muller, and R. Rothstein. 1998. A suppressor of two essential checkpoint genes identifies a novel protein that negatively affects dNTP pools. Mol. Cell 2, 329-340. https://doi.org/10.1016/S1097-2765(00)80277-4
  21. Zhao, X. and R. Rothstein. 2002. The Dun1 checkpoint kinase phosphorylates and regulates the ribonucleotide reductase inhibitor Sml1. Proc. Natl. Acad. Sci. USA 99, 3746-3751. https://doi.org/10.1073/pnas.062502299