The Effect of RAD7 Gene’s Null Mutation on Single-Cell Aging
Ismat Jahan Anee a, Reza Md Shahjahan a, Shamimul Alam a, Rowshan Ara Begum a, Md. Asaduzzaman Shishir b, Rakha Hari Sarker c and Ashfaqul Muid Khandaker a*
Microbial Bioactives 5(1) 171-180 https://doi.org/10.25163/microbbioacts.514307
Submitted: 09 March 2022 Revised: 30 May 2022 Published: 20 June 2022
Abstract
Background: Although different theories and hypotheses were postulated for aging, molecular mechanisms of its regulations are still vastly unknown. In time, a post-mitotic cell ages with the aggregation of mutations within its genome and reach senescence. The DNA repair system that protects its genome also malfunctions with time. This study aims to discover whether manipulating a DNA repair gene can regulate the cellular life span, especially the chronological life span using the single-cell model (Saccharomyces cerevisiae) in the field of aging. Methods: In this study, yeast mutant that lacks nucleotide excision repair (NER) gene RAD7 along with the diploid wild type (DP-WT) yeast strain (BY4743) as control were used. RAD7 encodes a protein that acts in the NER of UV-damaged DNA. To characterize the mutant cell, experiments i.e. chronological lifespan assay, growth-proliferation, respiration status, mtDNA distribution patterns, and interaction analysis using bioinformatics were conducted. MIPS functional classification using FunSpec software was used to determine RAD7’s interaction with diverse genes.Results: In the chronological lifespan assay, it was found that the RAD7 gene’s null mutation (Δrad7) had a prolonged stationary phase compared to the wild-type strain. The mutant displayed respiration potency by growing well on a glycerol-based medium. Fluorescence microscopic observations disclosed that the mutants had a lower abundance of mtDNA compared to the control. Furthermore, RAD7 gene interaction analysis demonstrated how it interacts with other genes in the organism. Mutation of this repair gene affects mtDNA distribution and mtDNA abundance, yet cells could survive in the chronological phase. MtDNA depletion is a sign of altered mitochondrial morphology and function that may activate a retrograde response from mitochondria to the nucleus. Conclusion: From this study, it is suggested that the RAD7 gene can regulate yeast cells’ life span. However, the retrograde response was not investigated in this current study and thus further efforts are also required to draw a fine conclusion.
Keywords: DNA Repair Gene; mitochondria; cell aging; RAD7; Saccharomyces cerevisiae.
References
Bilinski, Tomasz, Aneta Bylak, and Renata Zadrag-Tecza. 2017. "The Budding Yeast Saccharomyces Cerevisiae as a Model Organism: Possible Implications for Gerontological Studies." Biogerontology.
https://doi.org/10.1007/s10522-017-9712-x
PMid:28573416 PMCid:PMC5514200
Brown, Katharine. 2012. "Aging: The Mitochondrial Connection." Journal of Clinical & Experimental Pathology s4(01).
https://doi.org/10.4172/2161-0681.S4-003
Carmona-Gutierrez, Didac et al. 2018. "Guidelines and Recommendations on Yeast Cell Death Nomenclature." Microbial Cell.
Cone, Alan, and Alan Cone. 2018. "Yeast DAPI Staining." : 3-5.
Demir, Ayse Banu, and Ahmet Koc. 2010. "Assessment of Chronological Lifespan Dependent Molecular Damages in Yeast Lacking Mitochondrial Antioxidant Genes." Biochemical and Biophysical Research Communications 400(1): 106-10.
https://doi.org/10.1016/j.bbrc.2010.08.019
PMid:20707985
Denoth Lippuner, Annina, Thomas Julou, and Yves Barral. 2014. "Budding Yeast as a Model Organism to Study the Effects of Age." FEMS Microbiology Reviews 38(2): 300-325.
https://doi.org/10.1111/1574-6976.12060
PMid:24484434
Dodig, Slavica, Ivana Cepelak, and Ivan Pavic. 2019. "Hallmarks of Senescence and Aging." Biochemia Medica.
https://doi.org/10.11613/BM.2019.030501
PMid:31379458 PMCid:PMC6610675
Eisenberg, Tobias, and Sabrina Büttner. 2014. "Lipids and Cell Death in Yeast." FEMS Yeast Research.
https://doi.org/10.1111/1567-1364.12105
PMid:24119111 PMCid:PMC4255311
Emerit, J., M. Edeas, and F. Bricaire. 2004. "Neurodegenerative Diseases and Oxidative Stress." Biomedicine and Pharmacotherapy.
https://doi.org/10.1016/j.biopha.2003.11.004
PMid:14739060
Foury, Françoise. 1997. "Human Genetic Diseases: A Cross-Talk between Man and Yeast." Gene.
https://doi.org/10.1016/S0378-1119(97)00140-6
Friedberg, Errol C. et al. 2005. DNA Repair and Mutagenesis DNA Repair and Mutagenesis.
https://doi.org/10.1128/9781555816704
Game, John C., and Sophia B. Chernikova. 2009. "The Role of RAD6 in Recombinational Repair, Checkpoints and Meiosis via Histone Modification." DNA Repair.
https://doi.org/10.1016/j.dnarep.2009.01.007
PMid:19230796
Gangloff, Serge, and Benoit Arcangioli. 2017. "DNA Repair and Mutations during Quiescence in Yeast." FEMS Yeast Research 17(1): 1-14.
https://doi.org/10.1093/femsyr/fox002
PMid:28087675
Garcia, Enrique J. et al. 2019. "Reciprocal Interactions between MtDNA and Lifespan Control in Budding Yeast." Molecular Biology of the Cell.
https://doi.org/10.1091/mbc.E18-06-0356
PMid:31599702 PMCid:PMC6857569
Guzder, Sami N., Patrick Sung, Louise Prakash, and Satya Prakash. 1997a. "Yeast RAD7-RAD16 Complex, Specific for the Nucleotide Excision Repair of the Nontranscribed DNA Strand, Is an ATP-Dependent DNA Damage Sensor." Journal of Biological Chemistry.
https://doi.org/10.1074/jbc.272.35.21665
PMid:9268290
Guzder, Sami N., Patrick Sung, Louise Prakash, and Satya Prakash. 1997b. "Yeast RAD7-RAD16 Complex, Specific for the Nucleotide Excision Repair of the Nontranscribed DNA Strand, Is an ATP-Dependent DNA Damage Sensor." Journal of Biological Chemistry 272(35): 21665-68.
https://doi.org/10.1074/jbc.272.35.21665
PMid:9268290
Guzder, Sami N., Patrick Sung, Louise Prakash, and Satya Prakash. 1998. "The DNA-Dependent Atpase Activity of Yeast Nucleotide Excision Repair Factor 4 and Its Role in DNA Damage Recognition." Journal of Biological Chemistry.
Guzder, Sami N., Patrick Sung, Louise Prakash, and Satya Prakash. 1999. "Synergistic Interaction between Yeast Nucleotide Excision Repair Factors NEF2 and NEF4 in the Binding of Ultraviolet-Damaged DNA." Journal of Biological Chemistry.
Hacioglu, Elise, Ayse Banu Demir, and Ahmet Koc. 2012. "Identification of Respiratory Chain Gene Mutations That Shorten Replicative Life Span in Yeast." Experimental Gerontology.
https://doi.org/10.1016/j.exger.2011.11.009
PMid:22137892
Huh, Won Ki et al. 2003. "Global Analysis of Protein Localization in Budding Yeast." Nature.
https://doi.org/10.1038/nature02026
PMid:14562095
Hyman, Linda E. et al. 2002. "Binding to Elongin C Inhibits Degradation of Interacting Proteins in Yeast." Journal of Biological Chemistry.
https://doi.org/10.1074/jbc.M200800200
PMid:11864988
Janssens, Georges E., and Liesbeth M. Veenhoff. 2016. "Evidence for the Hallmarks of Human Aging in Replicatively Aging Yeast." Microbial Cell 3(7): 263-74.
https://doi.org/10.15698/mic2016.07.510
PMid:28357364 PMCid:PMC5354591
Jazwinski, S. Michal. 2014. "The Retrograde Response: A Conserved Compensatory Reaction to Damage from within and from Without." Progress in Molecular Biology and Translational Science 127: 133-54. https://doi.org/10.1016/B978-0-12-394625-6.00005-2
PMid:25149216 PMCid:PMC4271643
Kaeberlein, Matt. 2010. "Lessons on Longevity from Budding Yeast." Nature.
https://doi.org/10.1038/nature08981
PMid:20336133 PMCid:PMC3696189
Khandaker, Ashfaqul Muid, and Ahmet Koc. 2020. "Mitochondrial Targeted AFG3 Abolishment Triggers Higher Mitochondrial Membrane Potential (ΔΨm) in Young Yeast." Microbial Bioactives 3(1): 119-24. https://doi.org/10.25163/microbbioacts.31003A0713270620.
Khawaja, Abd Al Wahab, Zach R. Belak, Christopher H. Eskiw, and Troy A.A. Harkness. 2021. "High-Throughput Rapid Yeast Chronological Lifespan Assay." In Methods in Molecular Biology,.
https://doi.org/10.1007/978-1-0716-0868-5_18
PMid:32889725
Knorre, Dmitry A., Svyatoslav S. Sokolov, Anna N. Zyrina, and Fedor F. Severin. 2016. "How Do Yeast Sense Mitochondrial Dysfunction?" Microbial Cell.
https://doi.org/10.15698/mic2016.11.537
PMid:28357322 PMCid:PMC5349209
Kudryavtseva, Anna V. et al. 2016. "Mitochondrial Dysfunction and Oxidative Stress in Aging and Cancer." Oncotarget.
https://doi.org/10.18632/oncotarget.9821
PMid:27270647 PMCid:PMC5216692
Lahari, Triparna, Janelle Lazaro, Jeffrey M. Marcus, and Dana F. Schroeder. 2018. "RAD7 Homologues Contribute to Arabidopsis UV Tolerance." Plant Science 277(August): 267-77.
https://doi.org/10.1016/j.plantsci.2018.09.017
PMid:30466592
Lasserre, Jean Paul et al. 2015. "Yeast as a System for Modeling Mitochondrial Disease Mechanisms and Discovering Therapies." DMM Disease Models and Mechanisms.
https://doi.org/10.1242/dmm.020438
PMid:26035862 PMCid:PMC4457039
Lee, Sung-Keun, Sung-Lim Yu, Louise Prakash, and Satya Prakash. 2002. "Yeast RAD26, a Homolog of the Human CSB Gene, Functions Independently of Nucleotide Excision Repair and Base Excision Repair in Promoting Transcription through Damaged Bases." Molecular and Cellular Biology.
https://doi.org/10.1128/MCB.22.12.4383-4389.2002
PMid:12024048 PMCid:PMC133843
LeJeune, Danielle et al. 2009. "Yeast Elc1 Plays an Important Role in Global Genomic Repair but Not in Transcription Coupled Repair." DNA Repair.
https://doi.org/10.1096/fasebj.23.1_supplement.836.4
Lettieri, Teresa et al. 2008. "Functionally Distinct Nucleosome-Free Regions in Yeast Require Rad7 and Rad16 for Nucleotide Excision Repair." DNA Repair 7(5): 734-43.
https://doi.org/10.1016/j.dnarep.2008.01.016
PMid:18329964
Lombaerts, Marcel et al. 1999. "Characterization of the Rhp7+ and Rhp16+ Genes in Schizosaccharomyces Pombe." Nucleic Acids Research.
https://doi.org/10.1093/nar/27.17.3410
PMid:10446227 PMCid:PMC148581
Longo, V. D., and P. Fabrizio. 2002. "Regulation of Longevity and Stress Resistance: A Molecular Strategy Conserved from Yeast to Humans?" Cellular and Molecular Life Sciences.
https://doi.org/10.1007/s00018-002-8477-8
PMid:12169020
Longo, Valter D., Gerald S. Shadel, Matt Kaeberlein, and Brian Kennedy. 2012. "Replicative and Chronological Aging in Saccharomyces Cerevisiae." Cell Metabolism.
https://doi.org/10.1016/j.cmet.2012.06.002
PMid:22768836 PMCid:PMC3392685
López-Otín, Carlos et al. 2013. "The Hallmarks of Aging." Cell.
https://doi.org/10.1016/j.cell.2013.05.039
PMid:23746838 PMCid:PMC3836174
Marteijn, Jurgen A., Hannes Lans, Wim Vermeulen, and Jan H.J. Hoeijmakers. 2014. "Understanding Nucleotide Excision Repair and Its Roles in Cancer and Ageing." Nature Reviews Molecular Cell Biology.
https://doi.org/10.1038/nrm3822
PMid:24954209
Maynard, Scott et al. 2015. "DNA Damage, DNA Repair, Aging, and Neurodegeneration." Cold Spring Harbor Perspectives in Medicine.
https://doi.org/10.1101/cshperspect.a025130
PMid:26385091 PMCid:PMC4588127
Melis, Joost P.M., Harry Van Steeg, and Mirjam Luijten. 2013. "Oxidative DNA Damage and Nucleotide Excision Repair." Antioxidants and Redox Signaling.
https://doi.org/10.1089/ars.2012.5036
PMid:23216312 PMCid:PMC3671630
Michel, Jennifer J., Joseph F. McCarville, and Yue Xiong. 2003. "A Role for Saccharomyces Cerevisiae Cul8 Ubiquitin Ligase in Proper Anaphase Progression." Journal of Biological Chemistry.
https://doi.org/10.1074/jbc.M210358200
PMid:12676951
Moretti, Paolo, and David Shore. 2001. "Multiple Interactions in Sir Protein Recruitment by Rap1p at Silencers and Telomeres in Yeast." Molecular and Cellular Biology.
https://doi.org/10.1128/MCB.21.23.8082-8094.2001
PMid:11689698 PMCid:PMC99974
Muid, K. A. et al. 2019. "Characterization of Long Living Yeast Deletion Mutants That Lack Mitochondrial Metabolism Genes DSS1, PPA2 and AFG3." Gene 706(April): 172-80.
https://doi.org/10.1016/j.gene.2019.05.001
PMid:31082499
Ocampo, Alejandro et al. 2012. "Mitochondrial Respiratory Thresholds Regulate Yeast Chronological Life Span and Its Extension by Caloric Restriction." Cell Metabolism 16(1): 55-67.
https://doi.org/10.1016/j.cmet.2012.05.013
PMid:22768839 PMCid:PMC3397320
Ogata, Toshiya, Takanori Senoo, Shinji Kawano, and Shogo Ikeda. 2015. "Mitochondrial Superoxide Dismutase Deficiency Accelerates Chronological Aging in the Fission Yeast Schizosaccharomyces Pombe." Cell Biology International.
https://doi.org/10.1002/cbin.10556
PMid:26507459
Parrella, Edoardo, and Valter D. Longo. 2008. "The Chronological Life Span of Saccharomyces Cerevisiae to Study Mitochondrial Dysfunction and Disease." Methods.
https://doi.org/10.1016/j.ymeth.2008.10.004
PMid:18930829
Postnikoff, Spike D.L., Jay E. Johnson, and Jessica K. Tyler. 2017. "The Integrated Stress Response in Budding Yeast Lifespan Extension." Microbial Cell.
https://doi.org/10.15698/mic2017.11.597
PMid:29167799 PMCid:PMC5695854
Powell, C. D., D. E. Quain, and K. A. Smart. 2000. "The Impact of Media Composition and Petite Mutation on the Longevity of a Polyploid Brewing Yeast Strain." Letters in Applied Microbiology.
https://doi.org/10.1046/j.1472-765x.2000.00766.x
PMid:10886614
Prakash, Satya, and Louise Prakash. 2000. "Nucleotide Excision Repair in Yeast." Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis.
https://doi.org/10.1016/S0027-5107(00)00037-3
Prakash, Satya, Patrick Sung, and Louise Prakash. 1993. "DNA Repair Genes and Proteins of Saccharomyces Cerevisiae." Annual Review of Genetics.
https://doi.org/10.1146/annurev.ge.27.120193.000341
PMid:8122907
Reed, Simon H., Zhaoyang You, and Errol C. Friedberg. 1998. "The Yeast RAD7 and RAD16 Genes Are Required for Postincision Events during Nucleotide Excision Repair: In Vitro and in Vivo Studies with Rad7 and Rad16 Mutants and Purification of a Rad7/Rad16-Containing Protein Complex." Journal of Biological Chemistry 273(45): 29481-88.
https://doi.org/10.1074/jbc.273.45.29481
PMid:9792654
Rine, J., and I. Herskowitz. 1987. "Four Genes Responsible for a Position Effect on Expression from HML and HMR in Saccharomyces Cerevisiae." Genetics.
https://doi.org/10.1093/genetics/116.1.9
PMid:3297920 PMCid:PMC1203125
Schroeder, Elizabeth A., and Gerald S. Shadel. 2014. "Crosstalk between Mitochondrial Stress Signals Regulates Yeast Chronological Lifespan." Mechanisms of Ageing and Development.
https://doi.org/10.1016/j.mad.2013.12.002
PMid:24373996 PMCid:PMC3943709
Stefanini, Irene, Carlotta De Filippo, and Duccio Cavalieri. 2013. "Yeast as a Model in High-Throughput Screening of Small-Molecule Libraries." In Diversity-Oriented Synthesis,.
https://doi.org/10.1002/9781118618110.ch14
Svejstrup, Jesper Q. 2007. "Contending with Transcriptional Arrest during RNAPII Transcript Elongation." Trends in Biochemical Sciences.
https://doi.org/10.1016/j.tibs.2007.02.005
PMid:17349792
Swayne, Theresa C., Anna C. Gay, and Liza A. Pon. 2007. "Fluorescence Imaging of Mitochondria in Yeast." Methods in molecular biology (Clifton, N.J.) 372: 433-59.
https://doi.org/10.1007/978-1-59745-365-3_31
PMid:18314744
Tenreiro, Sandra, and Tiago Fleming Outeiro. 2010. "Simple Is Good: Yeast Models of Neurodegeneration." FEMS Yeast Research.
https://doi.org/10.1111/j.1567-1364.2010.00649.x
PMid:20579105
Tyrrell, Daniel J. et al. 2020. "Age-Associated Mitochondrial Dysfunction Accelerates Atherogenesis." Circulation Research.
https://doi.org/10.1161/CIRCRESAHA.119.315644
PMid:31818196 PMCid:PMC7006722
Walker, John M, S Eries E Ditor, and Plant Secondary Metabolites. Methods In Molecular Biology Tm.
Wallace, Douglas C. et al. 1998. "Mitochondrial Biology, Degenerative Diseases and Aging." In BioFactors,.
https://doi.org/10.1002/biof.5520070303
PMid:9568243
Wallace, Douglas C. et al. 2010. "Mitochondrial DNA Mutations in Disease and Aging." Environmental and Molecular Mutagenesis.
Waters, Raymond et al. 2012. "Nucleotide Excision Repair in Cellular Chromatin: Studies with Yeast from Nucleotide to Gene to Genome." International Journal of Molecular Sciences.
https://doi.org/10.3390/ijms130911141
PMid:23109843 PMCid:PMC3472735
Woo, Dong Kyun, and Robert O. Poyton. 2009. "The Absence of a Mitochondrial Genome in Rho0 Yeast Cells Extends Lifespan Independently of Retrograde Regulation." Experimental Gerontology.
https://doi.org/10.1016/j.exger.2009.03.001
PMid:19285548 PMCid:PMC3341797
Woudstra, Elies C. et al. 2002. "A Rad26-Def1 Complex Coordinates Repair and RNA Pol II Proteolysis in Response to DNA Damage." Nature.
https://doi.org/10.1038/415929a
PMid:11859374
Wu, Haiyan, and Anton J.M. Roks. 2014. "Genomic Instability and Vascular Aging: A Focus on Nucleotide Excision Repair." Trends in Cardiovascular Medicine.
https://doi.org/10.1016/j.tcm.2013.06.005
PMid:23953979
Zimmermann, Andreas et al. 2018. "Yeast as a Tool to Identify Anti-Aging Compounds." FEMS Yeast Research 18(6): 1-16.
https://doi.org/10.1093/femsyr/foy020
PMid:29905792 PMCid:PMC6001894
Zimmermann, A., Hofer, S., Pendl, T., Kainz, K., Madeo, F., Carmona-Gutierrez, D. (2018). Yeast as a tool to identify anti-aging compounds. FEMS Yeast Research, 18(6).
https://doi.org/10.1093/femsyr/foy020
PMid:29905792 PMCid:PMC6001894
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