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Rad7 E3 Ubiquitin Ligase Attenuates Polyubiquitylation of Rpn10 and Dsk2 Following DNA Damage in Saccharomyces cerevisiae

DOI: 10.4236/abc.2015.57021   PDF   HTML   XML   4,337 Downloads   4,869 Views  

Abstract

During Nucleotide Excision Repair (NER) in the yeast S. cerevisiae, ubiquitylation of Rad4 is carried out by the E3 ubiquitin ligase that includes Rad7-Elc1-Cul3 and is critical to optimal NER. Rad7 E3 activity targets Rad4 for degradation by the proteaseome but, in principle, could also trigger other DNA damage responses. We observed increased nuclear ubiquitin foci (Ub-RFP) formation in S. cerevisiae containing a Rad7 E3 ligase mutant (rad7SOCS) in response to DNA damage by benzo[a]pyrenediolepoxide (BPDE) in dividing cells. Immunoblots reveal that ubiquitin conjugates of Rpn10 and Dsk2 accumulate in greater abundance in rad7SOCS compared to RAD7 in dividing cells in response to BPDE which makes Rpn10 and Dsk2 candidates for being the ubiquitylated species observed in our microscopy experiments. Microscopy analysis with strains containing Dsk2-GFP shows that Dsk2-GFP and Dsk2-GFP/Ub-RFP colocalized in nuclear foci form to an increased extent in a rad7SOCS mutant background in dividing cells than in a RAD7 wild-type strain. Further, Dsk2-GFP in the rad7SOCS strain formed more foci at the plasma membrane following BPDE treatment in dividing cells relative to strains containing RAD7 or a rad7△ deletion mutant. In response to a different agent, UV irradiation, levels of ubiquitylated proteins were increased in rad7SOCS relative to RAD7, and the proteasomal deubiquitylase subunit, Rpn11 was even monoubiquitylated in the absence of damaging agents. Together these data show that Rad7 E3 activity attenuates ubiq-uitylation of proteins regulating the shuttling of polyubiquitylated proteins to the proteasome (Dsk2 and Rpn10) and removal of ubiquitin chains just prior to degradation (Rpn11). Since Rad7 E3 ligase activity has been shown to increase ubiquitylation of its target proteins, yet our results show increased ubiquitylation in the absence of Rad7 E3, we suggest that Rad7 E3 action regulates ubiquitin ligase and deubiquitylase (DUB) activities that act on Rpn10, Dsk2 and Rpn11.

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Benoun, J. , Lalimar-Cortez, D. , Valencia, A. , Granda, A. , Moore, D. , Kelson, E. and Fischhaber, P. (2015) Rad7 E3 Ubiquitin Ligase Attenuates Polyubiquitylation of Rpn10 and Dsk2 Following DNA Damage in Saccharomyces cerevisiae. Advances in Biological Chemistry, 5, 239-254. doi: 10.4236/abc.2015.57021.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1] Friedberg, E.C., Walker, G.C., Siede, W., Wood, R.D., Schultz, R. A., et al. (2005) DNA Repair and Mutagenesis. 2nd Edition, ASM Press, Washington DC.
[2] Staresincic, L., Fagbemi, A.F., Enzlin, J.H., Gourdin, A.M., Wijgers, N., et al. (2009) Coordination of Dual Incision and Repair Synthesis in Human Nucleotide Excision Repair. The EMBO Journal, 28, 1111-1120.
http://dx.doi.org/10.1038/emboj.2009.49
[3] Gillette, T.G., Yu, S., Zhou, Z., Waters, R., Johnston, S.A., et al. (2006) Distinct Functions of the Ubiquitin-Protea-some Pathway Influence Nucleotide Excision Repair. The EMBO Journal, 25, 2529-2538.
http://dx.doi.org/10.1038/sj.emboj.7601120
[4] Sugasawa, K., Okuda, Y., Saijo, M., Nishi, R., Matsuda, N., et al. (2005) UV-Induced Ubiquitylation of XPC Protein Mediated by UV-DDB-Ubiquitin Ligase Complex. Cell, 121, 387-400.
http://dx.doi.org/10.1016/j.cell.2005.02.035
[5] Teng, Y., Liu, H., Gill, H.W., Yu, Y., Waters, R., et al. (2008) Saccharomyces cerevisiae Rad16 Mediates Ultraviolet-Dependent Histone H3 Acetylation Required for Efficient Global Genome Nucleotide-Excision Repair. EMBO Reports, 9, 97-102.
http://dx.doi.org/10.1038/sj.embor.7401112
[6] Yu, S., Owen-Hughes, T., Friedberg, E.C., Waters, R. and Reed, S.H. (2004) The Yeast Rad7/Rad16/Abf1 Complex Generates Superhelical Torsion in DNA That Is Required for Nucleotide Excision Repair. DNA Repair, 3, 277-287.
http://dx.doi.org/10.1016/j.dnarep.2003.11.004
[7] Yu, S., Smirnova, J.B., Friedberg, E.C., Stillman, B., Akiyama, M., et al. (2009) ABF1-Binding Sites Promote Efficient Global Genome Nucleotide Excision Repair. The Journal of Biological Chemistry, 284, 966-973.
http://dx.doi.org/10.1074/jbc.M806830200
[8] Madura, K. (2004) Rad23 and Rpn10: Perennial Wallflowers Join the Melee. Trends in Biochemical Sciences, 29, 637- 640.
http://dx.doi.org/10.1016/j.tibs.2004.10.008
[9] Crosas, B., Hanna, J., Kirkpatrick, D.S., Zhang, D.P., Tone, Y., et al. (2006) Ubiquitin Chains Are Remodeled at the Proteasome by Opposing Ubiquitin Ligase and Deubiquitinating Activities. Cell, 127, 1401-1413.
http://dx.doi.org/10.1016/j.cell.2006.09.051
[10] Isasa, M., Katz, E.J., Kim, W., Yugo, V., Gonzalez, S., et al. (2010) Monoubiquitination of RPN10 Regulates Substrate Recruitment to the Proteasome. Molecular Cell, 38, 733-745.
http://dx.doi.org/10.1016/j.molcel.2010.05.001
[11] Mao, P. and Smerdon, M.J. (2010) Yeast Deubiquitinase Ubp3 Interacts with the 26 S Proteasome to Facilitate Rad4 Degradation. Journal of Biological Chemistry, 285, 37542-37550.
http://dx.doi.org/10.1074/jbc.M110.170175
[12] Liu, C., van Dyk, D., Li, Y., Andrews, B. and Rao, H. (2009) A Genome-Wide Synthetic Dosage Lethality Screen Reveals Multiple Pathways That Require the Functioning of Ubiquitin-Binding Proteins Rad23 and Dsk2. BMC Biology, 7, 75.
http://dx.doi.org/10.1186/1741-7007-7-75
[13] Finley, D. (2009) Recognition and Processing of Ubiquitin-Protein Conjugates by the Proteasome. Annual Review of Biochemistry, 78, 477-513.
http://dx.doi.org/10.1146/annurev.biochem.78.081507.101607
[14] Jaspersen, S.L. and Winey, M. (2004) The Budding Yeast Spindle Pole Body: Structure, Duplication, and Function. Annual Review of Cell and Developmental Biology, 20, 1-28.
http://dx.doi.org/10.1146/annurev.cellbio.20.022003.114106
[15] Diaz-Martinez, L.A., Kang, Y., Walters, K.J. and Clarke, D.J. (2006) Yeast UBL-UBA Proteins Have Partially Redundant Functions in Cell Cycle Control. Cell Division, 1, 28.
http://dx.doi.org/10.1186/1747-1028-1-28
[16] Kaye, F.J., Modi, S., Ivanovska, I., Koonin, E.V., Thress, K., et al. (2000) A Family of Ubiquitin-Like Proteins Binds the ATPase Domain of Hsp70-Like Stch. FEBS Letters, 467, 348-355.
http://dx.doi.org/10.1016/S0014-5793(00)01135-2
[17] Heessen, S., Masucci, M.G. and Dantuma, N.P. (2005) The UBA2 Domain Functions as an Intrinsic Stabilization Signal That Protects Rad23 from Proteasomal Degradation. Molecular Cell, 18, 225-235.
http://dx.doi.org/10.1016/j.molcel.2005.03.015
[18] Sekiguchi, T., Sasaki, T., Funakoshi, M., Ishii, T., Saitoh, Y.H., et al. (2011) Ubiquitin Chains in the Dsk2 UBL Domain Mediate Dsk2 Stability and Protein Degradation in Yeast. Biochemical and Biophysical Research Communications, 411, 555-561.
http://dx.doi.org/10.1016/j.bbrc.2011.06.183
[19] Zuin, A., Bichmann, A., Isasa, M., Puig-Sarries, P., Diaz, L.M., et al. (2015) Rpn10 Monoubiquitination Orchestrates the Association of the Ubiquilin-Type DSK2 Receptor with the Proteasome. Biochemical Journal, In Press.
http://dx.doi.org/10.1042/BJ20150609
[20] Lambertson, D., Chen, L. and Madura, K. (1999) Pleiotropic Defects Caused by Loss of the Proteasome-Interacting Factors Rad23 and Rpn10 of Saccharomyces cerevisiae. Genetics, 153, 69-79.
[21] Biggins, S., Ivanovska, I. and Rose, M.D. (1996) Yeast Ubiquitin-Like Genes Are Involved in Duplication of the Microtubule Organizing Center. Journal of Cell Biology, 133, 1331-1346.
http://dx.doi.org/10.1083/jcb.133.6.1331
[22] Liang, R.Y., Chen, L., Ko, B.T., Shen, Y.H., Li, Y.T., et al. (2014) Rad23 Interaction with the Proteasome Is Regulated by Phosphorylation of Its Ubiquitin-Like (UbL) Domain. Journal of Molecular Biology, 426, 4049-4060.
http://dx.doi.org/10.1016/j.jmb.2014.10.004
[23] Gillette, T.G., Huang, W., Russell, S.J., Reed, S.H., Johnston, S.A., et al. (2001) The 19S Complex of the Proteasome Regulates Nucleotide Excision Repair in Yeast. Genes & Development, 15, 1528-1539.
http://dx.doi.org/10.1101/gad.869601
[24] Li, Y., Yan, J., Kim, I., Liu, C., Huo, K., et al. (2010) Rad4 Regulates Protein Turnover at a Postubiquitylation Step. Molecular Biology of the Cell, 21, 177-185.
http://dx.doi.org/10.1091/mbc.E09-04-0305
[25] Lommel, L., Chen, L., Madura, K. and Sweder, K. (2000) The 26S Proteasome Negatively Regulates the Level of Overall Genomic Nucleotide Excision Repair. Nucleic Acids Research, 28, 4839-4845.
http://dx.doi.org/10.1093/nar/28.24.4839
[26] Lommel, L., Ortolan, T., Chen, L., Madura, K. and Sweder, K.S. (2002) Proteolysis of a Nucleotide Excision Repair Protein by the 26 S Proteasome. Current Genetics, 42, 9-20.
http://dx.doi.org/10.1007/s00294-002-0332-9
[27] Vizeacoumar, F.J., van Dyk, N., Vizeacoumar, F.S., Cheung, V., Li, J., et al. (2010) Integrating High-Throughput Genetic Interaction Mapping and High-Content Screening to Explore Yeast Spindle Morphogenesis. Journal of Cell Biology, 188, 69-81.
http://dx.doi.org/10.1083/jcb.200909013
[28] Miller, R.K., Cheng, S.C. and Rose, M.D. (2000) Bim1p/Yeb1p Mediates the Kar9p-Dependent Cortical Attachment of Cytoplasmic Microtubules. Molecular Biology of the Cell, 11, 2949-2959.
http://dx.doi.org/10.1091/mbc.11.9.2949
[29] Su, L.K., Burrell, M., Hill, D.E., Gyuris, J., Brent, R., et al. (1995) APC Binds to the Novel Protein EB1. Cancer Research, 55, 2972-2977.
[30] Rinaldi, T., Pick, E., Gambadoro, A., Zilli, S., Maytal-Kivity, V., et al. (2004) Participation of the Proteasomal Lid Subunit Rpn11 in Mitochondrial Morphology and Function Is Mapped to a Distinct C-Terminal Domain. Biochemical Journal, 381, 275-285.
http://dx.doi.org/10.1042/BJ20040008
[31] Bashore, C., Dambacher, C.M., Goodall, E.A., Matyskiela, M.E., Lander, G.C., et al. (2015) Ubp6 Deubiquitinase Controls Conformational Dynamics and Substrate Degradation of the 26S Proteasome. Nature Structural & Molecular Biology, 22, 712-719.
http://dx.doi.org/10.1038/nsmb.3075
[32] Aufderheide, A., Beck, F., Stengel, F., Hartwig, M., Schweitzer, A., et al. (2015) Structural Characterization of the Interaction of Ubp6 with the 26S Proteasome. Proceedings of the National Academy of Sciences of the United States of America, 112, 8626-8631.
http://dx.doi.org/10.1073/pnas.1510449112
[33] Novarina, D., Amara, F., Lazzaro, F., Plevani, P. and Muzi-Falconi, M. (2011) Mind the Gap: Keeping UV Lesions in Check. DNA Repair, 10, 751-759.
http://dx.doi.org/10.1016/j.dnarep.2011.04.030
[34] Campbell, R.E., Tour, O., Palmer, A.E., Steinbach, P.A., Baird, G.S., et al. (2002) A Monomeric Red Fluorescent Protein. Proceedings of the National Academy of Sciences of the United States of America, 99, 7877-7882.
http://dx.doi.org/10.1073/pnas.082243699
[35] Reid, R.J., Lisby, M. and Rothstein, R. (2002) Cloning-Free Genome Alterations in Saccharomyces cerevisiae Using Adaptamer-Mediated PCR. Methods in Enzymology, 350, 258-277.
http://dx.doi.org/10.1016/S0076-6879(02)50968-X
[36] Fleer, R., Nicolet, C.M., Pure, G.A. and Friedberg, E.C. (1987) RAD4 Gene of Saccharomyces cerevisiae: Molecular Cloning and Partial Characterization of a Gene That Is Inactivated in Escherichia coli. Molecular and Cellular Biology, 7, 1180-1192.
[37] Moore, D.M., Karlin, J., Gonzalez-Barrera, S., Mardiros, A., Lisby, M., et al. (2009) Rad10 Exhibits Lesion-Dependent Genetic Requirements for Recruitment to DNA Double-Strand Breaks in Saccharomyces cerevisiae. Nucleic Acids Research, 37, 6429-6438.
http://dx.doi.org/10.1093/nar/gkp709
[38] Huh, W.K., Falvo, J.V., Gerke, L.C., Carroll, A.S., Howson, R.W., et al. (2003) Global Analysis of Protein Localization in Budding Yeast. Nature, 425, 686-691.
[39] Mardiros, A., Benoun, J.M., Haughton, R., Baxter, K., Kelson, E.P., et al. (2011) Rad10-YFP Focus Induction in Response to UV Depends on RAD14 in Yeast. Acta Histochemica, 113, 409-415.
http://dx.doi.org/10.1016/j.acthis.2010.03.005
[40] Game, J.C. and Chernikova, S.B. (2009) The Role of RAD6 in Recombinational Repair, Checkpoints and Meiosis via Histone Modification. DNA Repair, 8, 470-482.
http://dx.doi.org/10.1016/j.dnarep.2009.01.007
[41] Weake, V.M. and Workman, J.L. (2008) Histone Ubiquitination: Triggering Gene Activity. Molecular Cell, 29, 653-663.
http://dx.doi.org/10.1016/j.molcel.2008.02.014
[42] Ulrich, H.D. (2005) Mutual Interactions between the SUMO and Ubiquitin Systems: A Plea of No Contest. Trends in Cell Biology, 15, 525-532.
http://dx.doi.org/10.1016/j.tcb.2005.08.002
[43] Ulrich, H.D. (2009) Regulating Post-Translational Modifications of the Eukaryotic Replication Clamp PCNA. DNA Repair, 8, 461-469.
http://dx.doi.org/10.1016/j.dnarep.2009.01.006
[44] Laporte, D., Salin, B., Daignan-Fornier, B. and Sagot, I. (2008) Reversible Cytoplasmic Localization of the Proteasome in Quiescent Yeast Cells. Journal of Cell Biology, 181, 737-745.
http://dx.doi.org/10.1083/jcb.200711154
[45] Funakoshi, M., Tomko Jr., R.J., Kobayashi, H. and Hochstrasser, M. (2009) Multiple Assembly Chaperones Govern Biogenesis of the Proteasome Regulatory Particle Base. Cell, 137, 887-899.
http://dx.doi.org/10.1016/j.cell.2009.04.061
[46] Seeger, M., Hartmann-Petersen, R., Wilkinson, C.R., Wallace, M., Samejima, I., et al. (2003) Interaction of the Anaphase-Promoting Complex/Cyclosome and Proteasome Protein Complexes with Multiubiquitin Chain-Binding Proteins. Journal of Biological Chemistry, 278, 16791-16796.
http://dx.doi.org/10.1074/jbc.M208281200
[47] Verhage, R.A., Van de Putte, P. and Brouwer, J. (1996) Repair of rDNA in Saccharomyces cerevisiae: RAD4-Independent Strand-Specific Nucleotide Excision Repair of RNA Polymerase I Transcribed Genes. Nucleic Acids Research, 24, 1020-1025.
http://dx.doi.org/5w0229
[48] Thomas, B.J. and Rothstein, R. (1989) Elevated Recombination Rates in Transcriptionally Active DNA. Cell, 56, 619- 630.
http://dx.doi.org/10.1016/0092-8674(89)90584-9

  
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