Characterization of the RAD52 Gene in the Budding Yeast Naumovozyma castellii
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains and Cell Culture
2.2. Transformation
2.3. Genotype Analysis of rad52Δ Transformants
2.4. Spot Assays
2.5. Survival Curves
2.6. Colony and Cell Imaging
2.7. Bioinformatic Analysis
3. Results and Discussion
3.1. DNA Sequence Analysis of the N. castellii RAD52 Gene
3.2. Deletion of the N. castellii RAD52 by Gene Replacement
3.3. Characterization of N. castellii rad52Δ Mutants
3.4. N. castellii rad52Δ Mutants Are Sensitive to Genotoxic Agents
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Symington, L.S. Mechanism and regulation of DNA end resection in eukaryotes. Crit. Rev. Biochem. Mol. Biol. 2016, 51, 195–212. [Google Scholar] [CrossRef] [PubMed]
- Llorente, B.; Smith, C.E.; Symington, L.S. Break-induced replication: What is it and what is it for? Cell Cycle 2008, 7, 859–864. [Google Scholar] [CrossRef]
- Lundblad, V. Telomere maintenance without telomerase. Oncogene 2002, 21, 522–531. [Google Scholar] [CrossRef]
- Mortensen, U.H.; Lisby, M.; Rothstein, R. Rad52. Curr. Biol. 2009, 19, R676–R677. [Google Scholar] [CrossRef]
- Symington, L.S. Role of RAD52 Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair. Microbiol. Mol. Biol. Rev. 2002, 66, 630. [Google Scholar] [CrossRef]
- San Filippo, J.; Sung, P.; Klein, H. Mechanism of eukaryotic homologous recombination. Annu. Rev. Biochem. 2008, 77, 229–257. [Google Scholar] [CrossRef] [PubMed]
- Symington, L.S.; Rothstein, R.; Lisby, M. Mechanisms and regulation of mitotic recombination in Saccharomyces cerevisiae. Genetics 2014, 198, 795–835. [Google Scholar] [CrossRef] [PubMed]
- Lundblad, V.; Blackburn, E.H. An alternative pathway for yeast telomere maintenance rescues est1− senescence. Cell 1993, 73, 347–360. [Google Scholar] [CrossRef]
- Chen, Q.; Ijpma, A.; Greider, C.W. Two survivor pathways that allow growth in the absence of telomerase are generated by distinct telomere recombination events. Mol. Cell. Biol. 2001, 21, 1819–1827. [Google Scholar] [CrossRef]
- Kagawa, W.; Kurumizaka, H.; Ishitani, R.; Fukai, S.; Nureki, O.; Shibata, T.; Yokoyama, S. Crystal Structure of the Homologous-Pairing Domain from the Human Rad52 Recombinase in the Undecameric Form. Mol. Cell 2002, 10, 359–371. [Google Scholar] [CrossRef]
- Nair, A.; Agarwal, R.; Chittela, R.K. Biochemical characterization of plant Rad52 protein from rice (Oryza sativa). Plant Physiol. Biochem. 2016, 106, 108–117. [Google Scholar] [CrossRef] [PubMed]
- Mortensen, U.H.; Erdeniz, N.; Feng, Q.; Rothstein, R. A molecular genetic dissection of the evolutionarily conserved N terminus of yeast Rad52. Genetics 2002, 161, 549–562. [Google Scholar] [CrossRef] [PubMed]
- Davis, A.P.; Symington, L.S. The Yeast Recombinational Repair Protein Rad59 Interacts With Rad52 and Stimulates Single-Strand Annealing. Genetics 2001, 159, 515–525. [Google Scholar] [CrossRef] [PubMed]
- Plate, I.; Hallwyl, S.C.L.; Shi, I.; Krejci, L.; Müller, C.; Albertsen, L.; Sung, P.; Mortensen, U.H. Interaction with RPA is necessary for Rad52 repair center formation and for its mediator activity. J. Biol. Chem. 2008, 283, 29077–29085. [Google Scholar] [CrossRef]
- Khade, N.V.; Sugiyama, T. Roles of C-terminal Region of Yeast and Human Rad52 in Rad51-Nucleoprotein Filament Formation and ssDNA Annealing. PLoS ONE 2016, 11, e0158436. [Google Scholar] [CrossRef]
- Karademir Andersson, A.; Oredsson, S.; Cohn, M. Development of stable haploid strains and molecular genetic tools for Naumovozyma castellii (Saccharomyces castellii). Yeast 2016, 33, 633–646. [Google Scholar] [CrossRef]
- Gordon, J.L.; Byrne, K.P.; Wolfe, K.H. Mechanisms of Chromosome Number Evolution in Yeast. PLoS Genet. 2011, 7, e1002190. [Google Scholar] [CrossRef]
- Kobayashi, N.; Suzuki, Y.; Schoenfeld, L.W.; Müller, C.A.; Nieduszynski, C.; Wolfe, K.H.; Tanaka, T.U. Discovery of an unconventional centromere in budding yeast redefines evolution of point centromeres. Curr. Biol. 2015, 25, 2026–2033. [Google Scholar] [CrossRef]
- Karademir Andersson, A.; Cohn, M. Naumovozyma castellii: An alternative model for budding yeast molecular biology. Yeast 2017, 34, 95–109. [Google Scholar] [CrossRef]
- Karademir Andersson, A.; Gustafsson, C.; Krishnankutty, R.; Cohn, M. Multiple DNA Interactions Contribute to the Initiation of Telomerase Elongation. J. Mol. Biol. 2017, 429, 2109–2123. [Google Scholar] [CrossRef]
- Reid, R.J.; Sunjevaric, I.; Keddache, M.; Rothstein, R. Efficient PCR-based gene disruption in Saccharomyces strains using intergenic primers. Yeast 2002, 19, 319–328. [Google Scholar] [CrossRef] [PubMed]
- Astromskas, E.; Cohn, M. Ends-in vs. ends-out targeted insertion mutagenesis in Saccharomyces castellii. Curr. Genet. 2009, 55, 339–347. [Google Scholar] [CrossRef] [PubMed]
- Kanehisa, M.; Goto, S. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 2000, 28, 27–30. [Google Scholar] [CrossRef] [PubMed]
- Cock, P.J.A.; Antao, T.; Chang, J.T.; Chapman, B.A.; Cox, C.J.; Dalke, A.; Friedberg, I.; Hamelryck, T.; Kauff, F.; Wilczynski, B.; et al. Biopython: Freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics 2009, 25, 1422–1423. [Google Scholar] [CrossRef]
- Iyer, L.M.; Koonin, E.V.; Aravind, L. Classification and evolutionary history of the single-strand annealing proteins, RecT, Redbeta, ERF and RAD52. BMC Genom. 2002, 3, 8. [Google Scholar] [CrossRef]
- Antúnez de Mayolo, A.; Lisby, M.; Erdeniz, N.; Thybo, T.; Mortensen, U.H.; Rothstein, R. Multiple start codons and phosphorylation result in discrete Rad52 protein species. Nucleic Acids Res. 2006, 34, 2587–2597. [Google Scholar] [CrossRef]
- Tsukamoto, M.; Yamashita, K.; Miyazaki, T.; Shinohara, M.; Shinohara, A. The N-terminal DNA-Binding Domain of Rad52 Promotes RAD51-Independent Recombination in Saccharomyces cerevisiae. Genetics 2003, 165, 1703–1715. [Google Scholar] [CrossRef]
- Lee, M.-S.; Yu, M.; Kim, K.-Y.; Park, G.-H.; Kwack, K.; Kim, K.P. Functional Validation of Rare Human Genetic Variants Involved in Homologous Recombination Using Saccharomyces cerevisiae. PLoS ONE 2015, 10, e0124152. [Google Scholar] [CrossRef]
- Davis, A.P.; Symington, L.S. The Rad52–Rad59 complex interacts with Rad51 and replication protein A. DNA Repair 2003, 2, 1127–1134. [Google Scholar] [CrossRef]
- Cortés-Ledesma, F.; Malagón, F.; Aguilera, A. A novel yeast mutation, rad52-L89F, causes a specific defect in Rad51-independent recombination that correlates with a reduced ability of Rad52-L89F to interact with Rad59. Genetics 2004, 168, 553–557. [Google Scholar] [CrossRef]
- Hanamshet, K.; Mazin, A.V. The function of RAD52 N-terminal domain is essential for viability of BRCA-deficient cells. Nucleic Acids Res. 2020, 48, 12778–12791. [Google Scholar] [CrossRef] [PubMed]
- Plate, I.; Albertsen, L.; Lisby, M.; Hallwyl, S.C.L.; Feng, Q.; Seong, C.; Rothstein, R.; Sung, P.; Mortensen, U.H. Rad52 multimerization is important for its nuclear localization in Saccharomyces cerevisiae. DNA Repair. 2008, 7, 57–66. [Google Scholar] [CrossRef] [PubMed]
- Sacher, M.; Pfander, B.; Hoege, C.; Jentsch, S. Control of Rad52 recombination activity by double-strand break-induced SUMO modification. Nat. Cell Biol. 2006, 8, 1284–1290. [Google Scholar] [CrossRef] [PubMed]
- Charifi, F.; Churikov, D.; Eckert-Boulet, N.; Minguet, C.; Jourquin, F.; Hardy, J.; Lisby, M.; Simon, M.-N.; Géli, V. Rad52 SUMOylation functions as a molecular switch that determines a balance between the Rad51- and Rad59-dependent survivors. iScience 2021, 24, 102231. [Google Scholar] [CrossRef]
- Krejci, L.; Song, B.; Bussen, W.; Rothstein, R.; Mortensen, U.H.; Sung, P. Interaction with Rad51 Is Indispensable for Recombination Mediator Function of Rad52. J. Biol. Chem. 2002, 277, 40132–40141. [Google Scholar] [CrossRef]
- Kagawa, W.; Arai, N.; Ichikawa, Y.; Saito, K.; Sugiyama, S.; Saotome, M.; Shibata, T.; Kurumizaka, H. Functional analyses of the C-terminal half of the Saccharomyces cerevisiae Rad52 protein. Nucleic Acids Res. 2014, 42, 941–951. [Google Scholar] [CrossRef]
- Bi, B.; Rybalchenko, N.; Golub, E.I.; Radding, C.M. Human and yeast Rad52 proteins promote DNA strand exchange. Proc. Natl. Acad. Sci. USA 2004, 101, 9568–9572. [Google Scholar] [CrossRef]
- Ranatunga, W.; Jackson, D.; Lloyd, J.A.; Forget, A.L.; Knight, K.L.; Borgstahl, G.E. Human RAD52 exhibits two modes of self-association. J. Biol. Chem. 2001, 276, 15876–15880. [Google Scholar] [CrossRef]
- Astromskas, E.; Cohn, M. Tools and methods for genetic analysis of Saccharomyces castellii. Yeast 2007, 24, 499–509. [Google Scholar] [CrossRef]
- Holland, C.L.; Weis, M.F.; England, C.J.; Berry, A.M.; Hall, P.D.; Lewis, L.K. Deficiency in homologous recombination is associated with changes in cell cycling and morphology in Saccharomyces cerevisiae. Exp. Cell Res. 2023, 430, 113701. [Google Scholar] [CrossRef]
- Yang, J.; Dungrawala, H.; Hua, H.; Manukyan, A.; Abraham, L.; Lane, W.; Mead, H.; Wright, J.; Schneider, B.L. Cell size and growth rate are major determinants of replicative lifespan. Cell Cycle 2011, 10, 144–155. [Google Scholar] [CrossRef] [PubMed]
- Marek, A.; Korona, R. Restricted pleiotropy facilitates mutational erosion of majot life-history traits. Evolution 2013, 67, 3077–3086. [Google Scholar] [CrossRef] [PubMed]
- Auesukaree, C.; Damnernsawad, A.; Kruatrachue, M.; Pokethitiyook, P.; Boonchird, C.; Kaneko, Y.; Harashima, S. Genome-wide identification of genes involved in tolerance to various environmental stresses in Saccharomyces cerevisiae. J. Appl. Genet. 2009, 50, 301–310. [Google Scholar] [CrossRef] [PubMed]
- Game, J.C.; Mortimer, R.K. A genetic study of x-ray sensitive mutants in yeast. Mutat. Res. 1974, 24, 281–292. [Google Scholar] [CrossRef]
- Chai, B.; Huang, J.; Cairns, B.R.; Laurent, B.C. Distinct roles for the RSC and Swi/Snf ATP-dependent chromatin remodelers in DNA double-strand break repair. Genes Dev. 2005, 19, 1656–1661. [Google Scholar] [CrossRef]
- Petermann, E.; Orta, M.L.; Issaeva, N.; Schultz, N.; Helleday, T. Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair. Mol. Cell 2010, 37, 492–502. [Google Scholar] [CrossRef] [PubMed]
- Xia, L.; Jaafar, L.; Cashikar, A.; Flores-Rozas, H. Identification of Genes Required for Protection from Doxorubicin by a Genome-Wide Screen in Saccharomyces cerevisiae. Cancer Res. 2007, 67, 11411–11418. [Google Scholar] [CrossRef]
- Harari, Y.; Zadok-Laviel, S.; Kupiec, M. Long Telomeres Do Not Affect Cellular Fitness in Yeast. mBio 2017, 8, e01314-17. [Google Scholar] [CrossRef]
- McKinney, J.S.; Sethi, S.; Tripp, J.D.; Nguyen, T.N.; A Sanderson, B.; Westmoreland, J.W.; Resnick, M.A.; Lewis, L.K. A multistep genomic screen identifies new genes required for repair of DNA double-strand breaks in Saccharomyces cerevisiae. BMC Genom. 2013, 14, 251. [Google Scholar] [CrossRef]
- Ngo, K.; Epum, E.A.; Friedman, K.L. Emerging non-canonical roles for the Rad51–Rad52 interaction in response to double-strand breaks in yeast. Curr. Genet. 2020, 66, 917–926. [Google Scholar] [CrossRef]
- Cohn, M.; Karademir Andersson, A.; Quintilla Mateo, R.; Carlsson Möller, M. Alternative Lengthening of Telomeres in the Budding Yeast Naumovozyma castellii. G3 Genes Genomes Genet. 2019, 9, 3345–3358. [Google Scholar] [CrossRef] [PubMed]
Strain | Genotype | Parental Strain | Reference |
---|---|---|---|
YMC48 | MATα, hoΔ::hphMX4, ura3 | YMC25 | Karademir Andersson et al., 2016 [16] |
YMC490-494 | MATα, hoΔ::hphMX4, ura3, rad52Δ::klURA3 | YMC48 | This study |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Itriago, H.; Marufee Islam, Z.; Cohn, M. Characterization of the RAD52 Gene in the Budding Yeast Naumovozyma castellii. Genes 2023, 14, 1908. https://doi.org/10.3390/genes14101908
Itriago H, Marufee Islam Z, Cohn M. Characterization of the RAD52 Gene in the Budding Yeast Naumovozyma castellii. Genes. 2023; 14(10):1908. https://doi.org/10.3390/genes14101908
Chicago/Turabian StyleItriago, Humberto, Zubaida Marufee Islam, and Marita Cohn. 2023. "Characterization of the RAD52 Gene in the Budding Yeast Naumovozyma castellii" Genes 14, no. 10: 1908. https://doi.org/10.3390/genes14101908
APA StyleItriago, H., Marufee Islam, Z., & Cohn, M. (2023). Characterization of the RAD52 Gene in the Budding Yeast Naumovozyma castellii. Genes, 14(10), 1908. https://doi.org/10.3390/genes14101908