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Cellular Dynamics of Rad51 and Rad54 in Response to Postreplicative Stress and DNA Damage in HeLa Cells

  • Choi, Eui-Hwan (Department of Life Sciences, Chung-Ang University) ;
  • Yoon, Seobin (Department of Life Sciences, Chung-Ang University) ;
  • Hahn, Yoonsoo (Department of Life Sciences, Chung-Ang University) ;
  • Kim, Keun P. (Department of Life Sciences, Chung-Ang University)
  • Received : 2016.11.13
  • Accepted : 2017.01.12
  • Published : 2017.02.28

Abstract

Homologous recombination (HR) is necessary for maintenance of genomic integrity and prevention of various mutations in tumor suppressor genes and proto-oncogenes. Rad51 and Rad54 are key HR factors that cope with replication stress and DNA breaks in eukaryotes. Rad51 binds to single-stranded DNA (ssDNA) to form the presynaptic filament that promotes a homology search and DNA strand exchange, and Rad54 stimulates the strand-pairing function of Rad51. Here, we studied the molecular dynamics of Rad51 and Rad54 during the cell cycle of HeLa cells. These cells constitutively express Rad51 and Rad54 throughout the entire cell cycle, and the formation of foci immediately increased in response to various types of DNA damage and replication stress, except for caffeine, which suppressed the Rad51-dependent HR pathway. Depletion of Rad51 caused severe defects in response to postreplicative stress. Accordingly, HeLa cells were arrested at the G2-M transition although a small amount of Rad51 was steadily maintained in HeLa cells. Our results suggest that cell cycle progression and proliferation of HeLa cells can be tightly controlled by the abundance of HR proteins, which are essential for the rapid response to postreplicative stress and DNA damage stress.

Keywords

DNA damage;HeLa cell;homologous recombination;Rad51;Rad54

Acknowledgement

Supported by : National Research Foundation of Korea

References

  1. Cejka, P., Cannavo, E., Polaczek, P., Masuda-Sasa, T., Pokharel, S., Campbell, J.L., and Kowalczykowski, S.C. (2010). DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2. Nature 467, 112-116. https://doi.org/10.1038/nature09355
  2. Chen, F., Nastasi, A., Shen, Z., Brenneman, M., Crissman, H., and Chen, D.J. (1997). Cell cycle-dependent protein expression of mammalian homologs of yeast DNA double-strand break repair genes Rad51 and Rad52. Mutat. Res. 384, 205-211. https://doi.org/10.1016/S0921-8777(97)00020-7
  3. Hong, S.G., Sung, Y.J., Yu, M., Lee, M.S., Kleckner, N., and Kim, K.P. (2013). The logic and mechanism of homologous recombination partner choice. Mol. Cell 51, 440-453. https://doi.org/10.1016/j.molcel.2013.08.008
  4. Jasin, M., and Rothstein, R. (2013). Repair of strand breaks by homologous recombination. Cold Spring Harb. Perspect Biol. 5, a012740. https://doi.org/10.1101/cshperspect.a012740
  5. Kim, K.P., Weiner, B.M., Zhang, L., Jordan, A., Dekker, J., and Kleckner, N. (2010). Sister cohesion and structural axis components mediate homolog bias of meiotic recombination. Cell 143, 924-937. https://doi.org/10.1016/j.cell.2010.11.015
  6. Blow, J.J., and Gillespie, P.J. (2008). Replication licensing and cancer-a fatal entanglement? Nat. Rev. Cancer 8, 799-806. https://doi.org/10.1038/nrc2500
  7. Branzei, D., and Foiani, M. (2010). Maintaining genome stability at the replication fork. Nat. Rev. Mol. Cell Biol. 11, 208-219. https://doi.org/10.1038/nrm2852
  8. Kim, D., Langmead, B., and Salzberg, S.L. (2015). HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357-360. https://doi.org/10.1038/nmeth.3317
  9. Krejci, L., Altmannova, V., Spirek, M., and Zhao, X. (2012). Homologous recombination and its regulation. Nucleic Acids Res. 40, 5795-5818. https://doi.org/10.1093/nar/gks270
  10. Lambert, S., and Lopez, B.S. (2000). Characterization of mammalian RAD51 double strand break repair using non-lethal dominantnegative forms. EMBO J. 19, 3090-3099. https://doi.org/10.1093/emboj/19.12.3090
  11. Lee, M.S., Yoon, S.W., and Kim, K.P. (2015). Mitotic cohesin subunit Mcd1 regulates the progression of meiotic recombination in budding yeast. J. Microbiol. Biotechnol. 25, 598-605 https://doi.org/10.4014/jmb.1501.01081
  12. Li, X., Zhang, X.P., Solinger, J.A., Kiianitsa, K., Yu, X., Egelman, E.H., and Heyer, W.D. (2007). Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics. Nucleic Acids Res.35, 4124-4140. https://doi.org/10.1093/nar/gkm412
  13. Mazin, A.V., Alexeev, A.A., and Kowalczykowski, S.C. (2003). A novel function of Rad54 protein. Stabilization of the Rad51 nucleoprotein filament. J. Biol. Chem., 278, 14029-14036. https://doi.org/10.1074/jbc.M212779200
  14. McGill, C.B., Shafer, B.K., Derr, L.K., and Strathern, J.N. (1993). Recombination initiated by double-strand breaks. Curr. Genet. 23, 305-314. https://doi.org/10.1007/BF00310891
  15. Merrick, C.J., Jackson, D., and Diffley, J.F. (2004). Visualization of altered replication dynamics after DNA damage in human cells. J. Biol. Chem. 279, 20067-20075. https://doi.org/10.1074/jbc.M400022200
  16. Nimonkar, A.V., Genschel, J., Kinoshita, E., Polaczek, P., Campbell, J.L., Wyman, C., Modrich, P., and Kowalczykowski, S.C. (2011). BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair. Genes Dev. 25, 350-362. https://doi.org/10.1101/gad.2003811
  17. Niu, H., Chung, W.H., Zhu, Z., Kwon, Y., Zhao, W., Chi, P., Prakash, R., Seong, C., Liu, D., Lu, L., et al. (2010). Mechanism of the ATPdependent DNA end-resection machinery from Saccharomyces cerevisiae. Nature 467, 108-111. https://doi.org/10.1038/nature09318
  18. Puchta, H., Dujon, B., and Hohn, B. (1993). Homologous recombination in plant cells is enhanced by in vivo induction of double strand breaks into DNA by a site-specific endonuclease. Nucleic Acids Res. 21, 5034-5040. https://doi.org/10.1093/nar/21.22.5034
  19. Ristic, D., Wyman, C., Paulusma, C., and Kanaar, R. (2001). The architecture of the human Rad54-DNA complex provides evidence for protein translocation along DNA. Proc. Natl. Acad. Sci. USA 15, 8454-8460.
  20. Rothstein, R., Michel, B., Gangloff, S. (2000). Replication fork pausing and recombination or "gimme a break". Genes Dev. 14, 1-10.
  21. Rouet, P., Smih, F.A., and Jasin, M. (1994). Introduction of doublestrand breaks into the genome of mouse cells by expression of a rarecutting endonuclease. Mol. Cell Biol. 14, 8096-8106. https://doi.org/10.1128/MCB.14.12.8096
  22. Sieber, O.M., Heinimann, K., and Tomlinson, I.P. (2003). Genomic instability-the engine of tumorigenesis? Nat. Rev. Cancer 3, 701-708. https://doi.org/10.1038/nrc1170
  23. Subramanyam, S., Ismail, M., Bhattacharya, I., and Spies, M. (2016). Tyrosine phosphorylation stimulates activity of human RAD51 recombinase through altered nucleoprotein filament dynamics. Proc. Natl. Acad. Sci. USA 10, 1073-1082.
  24. Yoon, S.W., Kim, D.K., Kim, K.P., and Park, K.S. (2014). Rad51 regulates cell cycle progression by preserving G2/M transition in mouse embryonic stem cells. Stem Cells Dev. 23, 2700-2711. https://doi.org/10.1089/scd.2014.0129
  25. Yoon, S.W., Lee, M.S., Xaver, M., Zhang, L., Hong, S.G., Kong, Y.J., Cho, H.R., Kleckner, N., and Kim, K.P. (2016). Meiotic prophase roles of Rec8 in crossover recombination and chromosome structure. Nucleic Acids Res. 44, 9296-9314.
  26. Zelensky, A.N., Sanchez, H., Ristic, D., Vidic, I., van Rossum-Fikkert, S.E., Essers, J., Wyman, C., and Kanaar, R. (2013). Caffeine suppresses homologous recombination through interference with RAD51-mediated joint molecule formation. Nucleic Acids Res. 41, 6475-6489. https://doi.org/10.1093/nar/gkt375
  27. Zhdanova, N.S., and Rubtsov, N.B. (2016). Telomere recombination in normal mammalian cells. Genetika 54, 14-23.

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