DOI QR코드

DOI QR Code

효모에서 Hrq1과 Rad14의 상호작용에 대한 연구

Characterization of Hrq1-Rad14 Interaction in Saccharomyces cerevisiae

  • 민문희 (인하대학교 자연과학대학 생명과학과) ;
  • 김민지 (인하대학교 자연과학대학 생명과학과) ;
  • 최유진 (학익여자고등학교) ;
  • 유민주 (학익여자고등학교) ;
  • 김유라 (학익여자고등학교) ;
  • 안효빈 (학익여자고등학교) ;
  • 김채현 (학익여자고등학교) ;
  • 권채연 (학익여자고등학교) ;
  • 배성호 (인하대학교 자연과학대학 생명과학과)
  • 투고 : 2014.03.24
  • 심사 : 2014.05.29
  • 발행 : 2014.06.30

초록

Hrq1은 곰팡이 유전체에서 생물정보분석에 의해 발견된 새로운 RecQ helicase이다. 이 단백질은 인간의 RECQL4와 가장 상동성이 높으며 최근의 유전학적 생화학적 연구를 통해서 유전체 안정성을 유지하는데 어떤 역할을 할 것으로 예상되었다. 본 연구에서는 RECQL4와 상호작용하는 것으로 알려진 인간 유전자들과 상동성이 있는 효모 유전자들이 Hrq1과 상호작용하는지를 yeast two-hybrid assay를 이용하여 조사하였다. 총 11개의 유전자를 조사한 결과, nucleotide excision repair (NER) 인자 중의 하나인 Rad14이 Hrq1과 상호작용하는 것을 발견하였다. 또한 정제한 단백질을 이용한 pull-down assay로 Hrq1과 Rad14 사이의 직접적인 상호작용을 확인하였다. Hrq1과 Rad14 사이의 yeast two-hybrid 상호작용은 4-nitroquinoline-1-oxide에 의한 DNA 손상으로 더욱 증가하였으며, 이러한 상호작용의 증가는 또 다른 NER 인자인 Rad4에 의존적이었다. 이러한 결과들은 Hrq1이 Rad14과의 상호작용을 통하여 NER 과정에 어떤 역할을 할 가능성을 제시하고 있다.

Hrq1 is a novel member of RecQ helicase family, found in fungal genomes by bioinformatics analyses. It is most homologous to human RECQL4 and recent genetic and biochemical studies suggested that it may play roles in the maintenance of genome stability. In this study, we investigated yeast two-hybrid interactions between Hrq1 and the yeast genes homologous to the human genes that are known to interact with RECQL4. Among the 11 genes tested, Rad14, a nucleotide excision repair (NER) factor, was found to interact with Hrq1. In addition, pull-down assay with the purified proteins revealed direct protein-protein interaction between Hrq1 and Rad14. The yeast two-hybrid interaction was enhanced by the DNA damage induced by 4-nitroquinoline-1-oxide, which was dependent on the presence of Rad4, a key NER factor. These results suggest that Hrq1 may function in NER through interaction with Rad14.

키워드

참고문헌

  1. Ashton, T.M. and Hickson, I.D. 2010. Yeast as a model system to study RecQ helicase function. DNA Repair 9, 303-314. https://doi.org/10.1016/j.dnarep.2009.12.007
  2. Barea, F., Tessaro, S., and Bonatto, D. 2008. In silico analyses of a new group of fungal and plant RecQ4-homologous proteins. Comput. Biol. Chem. 32, 349-358. https://doi.org/10.1016/j.compbiolchem.2008.07.005
  3. Baudin, A., Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F., and Cullin, C. 1993. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 21, 3329-3330. https://doi.org/10.1093/nar/21.14.3329
  4. Bernstein, K.A., Gangloff, S., and Rothstein, R. 2010. The RecQ DNA helicases in DNA repair. Annu. Rev. Genet. 44, 393-417. https://doi.org/10.1146/annurev-genet-102209-163602
  5. Bohr, V.A. 2008. Rising from the RecQ-age: the role of human RecQ helicases in genome maintenance. Trends Biochem. Sci. 33, 609-620. https://doi.org/10.1016/j.tibs.2008.09.003
  6. Choi, D.H., Lee, R., Kwon, S.H., and Bae, S.H. 2013. Hrq1 functions independently of Sgs1 to preserve genome integrity in Saccharomyces cerevisiae. J. Microbiol. 51, 105-112. https://doi.org/10.1007/s12275-013-3048-2
  7. Choi, D.H., Min, M.H., Kim, M.J., Lee, R., Kwon, S.H., and Bae, S.H. 2014. Hrq1 facilitates nucleotide excision repair of DNA damage induced by 4-nitroquinoline-1-oxide and cisplatin in Saccharomyces cerevisiae. J. Microbiol. 52, 292-298. https://doi.org/10.1007/s12275-014-4018-z
  8. Chris, K., Michaelis, S., and Mitchell, A. 1994. Methods in yeast genetics. pp. 207-217. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, N.Y., USA.
  9. Chu, W.K. and Hickson, I.D. 2009. RecQ helicases: multifunctional genome caretakers. Nat. Rev. Cancer 9, 644-654. https://doi.org/10.1038/nrc2682
  10. Compe, E. and Egly, J.M. 2012. TFIIH: when transcription met DNA repair. Nat. Rev. Mol. Cell Biol. 13, 343-354. https://doi.org/10.1038/nrm3350
  11. Croteau, D.L., Singh, D.K., Ferrarelli, L.H., Lu, H., and Bohr, V.A. 2012. RECQL4 in genomic instability and aging. Trends Genet. 28, 624-631. https://doi.org/10.1016/j.tig.2012.08.003
  12. Fan, W. and Luo, J. 2008. RecQ4 facilitates UV light-induced DNA damage repair through interaction with nucleotide excision repair factor xeroderma pigmentosum group A (XPA). J. Biol. Chem. 283, 29037-29044. https://doi.org/10.1074/jbc.M801928200
  13. Groocock, L.M., Prudden, J., Perry, J.J., and Boddy, M.N. 2012. The RecQ4 orthologue Hrq1 is critical for DNA interstrand cross-link repair and genome stability in fission yeast. Mol. Cell Biol. 32, 276-287. https://doi.org/10.1128/MCB.06184-11
  14. Guzder, S.N., Sommers, C.H., Prakash, L., and Prakash, S. 2006. Complex formation with damage recognition protein Rad14 is essential for Saccharomyces cerevisiae Rad1-Rad10 nuclease to perform its function in nucleotide excision repair in vivo. Mol. Cell Biol. 26, 1135-1141. https://doi.org/10.1128/MCB.26.3.1135-1141.2006
  15. Huang, M. and Elledge, S.J. 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
  16. James, P., Halladay, J., and Craig, E.A. 1996. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144, 1425-1436.
  17. Kwon, S.H., Choi, D.H., Lee, R., and Bae, S.H. 2012. Saccharomyces cerevisiae Hrq1 requires a long 3'-tailed DNA substrate for helicase activity. Biochem. Biophys. Res. Commun. 427, 623-628. https://doi.org/10.1016/j.bbrc.2012.09.109
  18. Lafrance-Vanasse, J., Arseneault, G., Cappadocia, L., Legault, P., and Omichinski, J.G. 2013. Structural and functional evidence that Rad4 competes with Rad2 for binding to the Tfb1 subunit of TFIIH in NER. Nucleic Acids Res. 41, 2736-2745. https://doi.org/10.1093/nar/gks1321
  19. Mardiros, A., Benoun, J.M., Haughton, R., Baxter, K., Kelson, E.P., and Fischhaber, P.L. 2011. Rad10-YFP focus induction in response to UV depends on RAD14 in yeast. Acta Histochem. 113, 409-415. https://doi.org/10.1016/j.acthis.2010.03.005
  20. Prakash, S. and Prakash, L. 2000. Nucleotide excision repair in yeast. Mutat. Res. 451, 13-24. https://doi.org/10.1016/S0027-5107(00)00037-3
  21. Riedl, T., Hanaoka, F., and Egly, J.M. 2003. The comings and goings of nucleotide excision repair factors on damaged DNA. EMBO J. 22, 5293-5303. https://doi.org/10.1093/emboj/cdg489
  22. Rodriguez, K., Talamantez, J., Huang, W., Reed, S.H., Wang, Z., Chen, L., Feaver, W.J., Friedberg, E.C., and Tomkinson, A.E. 1998. Affinity purification and partial characterization of a yeast multiprotein complex for nucleotide excision repair using histidine-tagged Rad14 protein. J. Biol. Chem. 273, 34180-34189. https://doi.org/10.1074/jbc.273.51.34180
  23. Tsodikov, O.V., Ivanov, D., Orelli, B., Staresincic, L., Shoshani, I., Oberman, R., Scharer, O.D., Wagner, G., and Ellenberger, T. 2007. Structural basis for the recruitment of ERCC1-XPF to nucleotide excision repair complexes by XPA. EMBO J. 26, 4768-4776. https://doi.org/10.1038/sj.emboj.7601894
  24. Wang P.J., Chabes, A., Casagrande, R., Tian, X.C., Thelander, L., and Huffaker, T.C. 1997. Rnr4p, a novel ribonucleotide reductase small-subunit protein. Mol. Cell Biol. 17, 6114-6121. https://doi.org/10.1128/MCB.17.10.6114