In vivo and in silico analysis of PCNA ubiquitylation in the activation of the Post Replication Repair pathway in S. cerevisiae

BMC Systems Biology

Paolo Plevani⁵  Daniela Besozzi³  Marco Muzi Falconi⁵  Attila Csikász-Nagy⁴  Dario Pescini¹  Paolo Cazzaniga²  Riccardo Colombo⁶  Flavio Amara⁵ 

[1] Dipartimento di Statistica e Metodi Quantitativi, Università degli Studi di Milano-Bicocca, Milano, Italy;Dipartimento di Scienze Umane e Sociali, Università degli Studi di Bergamo, Bergamo, Italy;Dipartimento di Informatica, Università degli Studi di Milano, Milano, Italy;, The Microsoft Research - Università degli Studi di Trento, Centre for Computational and Systems Biology, Rovereto (Trento), Italy;Dipartimento di Bioscienze, Università degli Studi di Milano, Milano, Italy;Dipartimento di Informatica, Sistemistica e Comunicazione, Università degli Studi di Milano-Bicocca, Milano, Italy

关键词: Paramater sweep analysis;    Stochastic simulation;    Mechanistic modeling;    Budding yeast;    Ubiquitylation;    PCNA;    Post replication repair;    DNA damage;   

Others  :  1143030
DOI  :  10.1186/1752-0509-7-24

 received in 2012-07-24, accepted in 2013-02-05,  发布年份 2013

【 摘 要 】

Background

The genome of living organisms is constantly exposed to several damaging agents that induce different types of DNA lesions, leading to cellular malfunctioning and onset of many diseases. To maintain genome stability, cells developed various repair and tolerance systems to counteract the effects of DNA damage. Here we focus on Post Replication Repair (PRR), the pathway involved in the bypass of DNA lesions induced by sunlight exposure and UV radiation. PRR acts through two different mechanisms, activated by mono- and poly-ubiquitylation of the DNA sliding clamp, called Proliferating Cell Nuclear Antigen (PCNA).

Results

We developed a novel protocol to measure the time-course ratios between mono-, di- and tri-ubiquitylated PCNA isoforms on a single western blot, which were used as the wet readout for PRR events in wild type and mutant S. cerevisiae cells exposed to acute UV radiation doses. Stochastic simulations of PCNA ubiquitylation dynamics, performed by exploiting a novel mechanistic model of PRR, well fitted the experimental data at low UV doses, but evidenced divergent behaviors at high UV doses, thus driving the design of further experiments to verify new hypothesis on the functioning of PRR. The model predicted the existence of a UV dose threshold for the proper functioning of the PRR model, and highlighted an overlapping effect of Nucleotide Excision Repair (the pathway effectively responsible to clean the genome from UV lesions) on the dynamics of PCNA ubiquitylation in different phases of the cell cycle. In addition, we showed that ubiquitin concentration can affect the rate of PCNA ubiquitylation in PRR, offering a possible explanation to the DNA damage sensitivity of yeast strains lacking deubiquitylating enzymes.

Conclusions

We exploited an in vivo and in silico combinational approach to analyze for the first time in a Systems Biology context the events of PCNA ubiquitylation occurring in PRR in budding yeast cells. Our findings highlighted an intricate functional crosstalk between PRR and other events controlling genome stability, and evidenced that PRR is more complicated and still far less characterized than previously thought.

【 授权许可】

   
2013 Amara et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Hoeijmakers J: Genome maintenance mechanisms for preventing cancer. Nature 2001, 411:366-374.
• [2]Rolig RL, McKinnon PJ: Linking DNA damage and neurodegeneration. Trends Neurosci 2000, 23:417-424.
• [3]Moldovan G, Pfander B, Jentsch S: PCNA, the maestro of the replication fork. Cell 2007, 129(4):665-679.
• [4]Sinha RP, Hader DP: UV-induced DNA damage and repair: a review. Photochem Photobiol Sci 2002, 1:225-236.
• [5]Mitchell D, Karentz D: The induction and repair of DNA photodamage in the environment. In Environmental UV Photobiology. Edited by Young A, Bjorn L, Moan J, Nultsch W. New York: Plenum; 1993:345-377.
• [6]Friedberg E, Walker G, Siede W: DNA Repair and Mutagenesis. Washington: ASM Press; 1995.
• [7]Lazzaro F, Giannattasio M, Puddu F, Granata M, Pellicioli A, Plevani P, Muzi-Falconi M: Checkpoint mechanisms at the intersection between DNA damage and repair. DNA Repair 2009, 8(9):1055-1067.
• [8]Novarina D, Amara F, Lazzaro F, Plevani P, Muzi-Falconi M: Mind the gap: Keeping UV lesions in check. DNA Repair 2011, 10(7):751-759.
• [9]Ulrich HD, Walden H: Ubiquitin signalling in DNA replication and repair. Nat Rev Mol Cell Biol 2010, 11(7):479-489.
• [10]Pan X, Ye P, Yuan DS, Wang X, Bader JS, Boeke JD: A DNA integrity network in the yeast Saccharomyces cerevisiae. Cell 2006, 124(5):1069-1081.
• [11]Lisby M, Barlow JH, Burgess RC, Rothstein R: Choreography of the DNA damage response. Cell 2004, 118(6):699-713.
• [12]Milanowska K, Rother K, Bujnicki J: Databases and bioinformatics tools for the study of DNA repair. Mol Biol Int 2011, 2011:Article ID 475718.
• [13]Qu Z, MacLellan WR, Weiss JN: Dynamics of the cell cycle: checkpoints, sizers, and timers. Biophys J 2003, 85(6):3600-3611.
• [14]Iwamoto K, Tashima Y, Hamada H, Eguchi Y, Okamoto M: Mathematical modeling and sensitivity analysis of G1/S phase in the cell cycle including the DNA-damage signal transduction pathway. BioSystems 2008, 94(1-2):109-117.
• [15]Iwamoto K, Hamada H, Eguchi Y, Okamoto M: Mathematical modeling of cell cycle regulation in response to DNA damage: Exploring mechanisms of cell-fate determination. BioSystems 2011, 103(3):384-391.
• [16]Csikász-Nagy A: Computational systems biology of the cell cycle. Brief Bioinform 2009, 10(4):424-434.
• [17]Karschau J, de Almeida C, Richard MC, Miller S, Booth IR, Grebogi C, de Moura AP: A matter of life or death: modeling DNA damage and repair in bacteria. Biophys J 2011, 100(4):814-821.
• [18]Krishna S, Maslov S, Sneppen K: UV-induced mutagenesis in Escherichia coli SOS response: A quantitative model. PLoS Comput Biol 2007, 3(3):e41.
• [19]Mouri K, Nacher J, Akutsu T: A mathematical model for the detection mechanism of DNA double-strand breaks depending on autophosphorylation of ATM. PLoS ONE 2009, 4(4):e5131.
• [20]Crooke PS, Parl FF: A mathematical model for DNA damage and repair. J Nucleic Acids 2010, 2010:Article ID 352603.
• [21]Sokhansanj BA, Rodrigue GR, Fitch JP, Wilson III DM: A quantitative model of human DNA base excision repair. I. mechanistic insights. Nucleic Acids Res 2002, 30(8):1817-1825.
• [22]Politi A, Moné MJ, Houtsmuller AB, Hoogstraten D, Vermeulen W, Heinrich R, van Driel R: Mathematical modeling of nucleotide excision repair reveals efficiency of sequential assembly strategies. Mol Cell 2005, 19(5):679-690.
• [23]Kesseler KJ, Kaufmann WK, Reardon JT, Elston T, Sancar A: A mathematical model for human nucleotide excision repair: damage recognition by random order assembly and kinetic proofreading. J Theor Biol 2007, 249(2):361-375.
• [24]Luijsterburg MS, von Bornstaedt G, Gourdin AM, Politi AZ, Moné MJ, Warmerdam DO, Goedhart J, Vermeulen W, van Driel R, Höffer T: Stochastic and reversible assembly of a multiprotein DNA repair complex ensures accurate target site recognition and efficient repair. J Cell Biol 2010, 189(3):445-463.
• [25]Hoege C, Pfander B, Moldovan G, Pyrowolakis G, Jentsch S: RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 2002, 419:135-141.
• [26]Zhang W, Qin Z, Zhang X, Xiao W: Roles of sequential ubiquitination of PCNA in DNA-damage tolerance. FEBS Lett 2011, 585(18):2786-2794.
• [27]Daigaku Y, Davies AA, Ulrich HD: Ubiquitin-dependent DNA damage bypass is separable from genome replication. Nature 2010, 465(7300):951-955.
• [28]Gillespie DT: Exact stochastic simulation of coupled chemical reactions. J Phys Chem 1977, 81(25):2340-2361.
• [29]Neecke H, Lucchini G, Longhese M: Cell cycle progression in the presence of irreparable DNA damage is controlled by a Mec1- and Rad53-dependent checkpoint in budding yeast. EMBO J 1999, 18(16):4485-97.
• [30]Gong J, Siede W: Influence of deubiquitinating enzymes on mutagenesis in Saccharomyces cerevisiae. Internet J Microb 2011., 9(2)
• [31]Zhao S, Ulrich H: Distinct consequences of posttranslational modification by linear versus K63-linked polyubiquitin chain. P Natl Acad Sci USA 2010, 107(17):7704-7709.
• [32]Essers J, Theil AF, Baldeyron C, van Cappellen WA, Houtsmuller AB, Kanaar R, Vermeulen W: Nuclear dynamics of PCNA in DNA replication and repair. Mol Cell Biol 2005, 25(21):9350-9359.
• [33]Haas A, Rose I: The mechanism of ubiquitin activating enzyme. A kinetic and equilibrium analysis. J Biol Chem 1982, 257(17):10329-10337.
• [34]Hofmann RM, Pickart CM: Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell 1999, 96:645-653.
• [35]Vandemark AP, Hofmann RM, Tsui C, Pickart CM, Wolberger C: Molecular insights into polyubiquitin chain assembly: crystal structure of the Mms2/Ubc13 heterodimer. Structure 2001, 105:711-720.
• [36]Parker J, Ulrich HD: Mechanistic analysis of PCNA poly-ubiquitylation by the ubiquitin protein ligases Rad18 and Rad5. EMBO J 2009, 28:3657-3666.
• [37]Davies A, Huttner D, Daigaku Y, Chen S, HD HU: Activation of ubiquitin-dependent DNA damage bypass is mediated by replication protein A. Mol Cell 2008, 29(5):625-636.
• [38]Windecker H, Ulrich HD: Architecture and assembly of poly-SUMO Chains on PCNA in Saccharomyces cerevisiae. J Mol Biol 2008, 376:221-231.
• [39]Friedberg E, Lehmann A, Fuchs R: Trading places: how do DNA polymerases switch during translesion DNA synthesis? Mol Cell 2005, 18:499-505.
• [40]Johnson RE, Haracska L, Prakash S, Prakash L: Role of DNA polymerase η in the bypass of a (6-4) TT photoproduct. Mol Cell Biol 2001, 21(10):3558-3563.
• [41]Nelson JR, Lawrence CW, Hinkle DC: Thymine-thymine dimer bypass by yeast DNA polymerase. Science 1996, 272:1646-1649.
• [42]Longtine MS, Mckenzie III A, Demarini DJ, Shah NG, Wach A, Brachat A, Philippsen P, Pringle JR: Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 1998, 14(10):953-961.
• [43]Longhese M, Fraschini R, Plevani P, Lucchini G: Yeast pip3/mec3 mutants fail to delay entry into S phase and to slow DNA replication in response to DNA damage, and they define a functional link between Mec3 and DNA primase. Mol Cell Biol 1996, 16(7):3235-3244.
• [44]Hanna J, Leggett D, Finley D: Ubiquitin depletion as a key mediator of toxicity by translational inhibitors. Mol Cell Biol 2003, 23(24):9251-9261.
• [45]Lazzaro F, Novarina D, Amara F, Watt DL, Stone JE, Costanzo V, Burgers PM, Kunkel TA, Plevani P, Muzi-Falconi M: RNase H and Postreplication Repair protect cells from ribonucleotides incorporated in DNA. Mol Cell 2012, 45:99-110.
• [46]Ulrich HD, Davies AA: In vivo detection and characterization of sumoylation targets in Saccharomyces cerevisiae. Methods Mol Biol 2009, 497(II):81-103.
• [47]Hafiz A: Principles and Reactions of Protein Extraction, Purification, and Characterization. Boca Raton: CRC Press, Taylor and Francis Group; 2004.
• [48]Abramoff M, Magelhaes P, Ram S: Image processing with ImageJ. Biophotonics Int 2004, 11(7):36-42.
• [49]Ghaemmaghami S, Huh W, Bower K, Howson R, Belle A, Dephoure N, et al.: Global analysis of protein expression in yeast. Nature 2003, 425(6959):671-672.
• [50]Cherry JM, Hong EL, Amundsen C, Balakrishnan R, Binkley G, Chan ET, Christie KR, Costanzo MC, Dwight SS, Engel SR, Fisk DG, Hirschman JE, Hitz BC, Karra K, Krieger CJ, Miyasato SR, Nash RS, Park J, Skrzypek MS, Simison M, Weng S, Wong ED: Saccharomyces Genome Database: the genomics resource of budding yeast. Nucleic Acids Res Jan 2012, 40(Database issue):D700—D705.
• [51]SGD project: Saccharomyces Genome Database. 2011. [http://www.yeastgenome.org webcite]
• [52]VonDerHaar T: A quantitative estimation of the global translational activity in logarithmically growing yeast cells. BMC Syst Biol 2008., 2(87)
• [53]Jorgensen P, Edgington N, Schneider B, Rupes I, Tyers M, Futcher B: The size of the nucleus increases as yeast cells grow. Mol Biol Cell 2007, 18:3523-3532.
• [54]Carlile CM, Pickart CM, Matunis MJ, Cohen RE: Synthesis of free and Proliferating Cell Nuclear Antigen-bound polyubiquitin chains by the RING E3 ubiquitin ligase Rad5. J Biol Chem 2009, 284(43):29326-29334.
• [55]Notenboom V, Hibbert RG, van Rossum-Fikkert SE, Olsen JV, Mann M, Sixma TK: Functional characterization of Rad18 domains for Rad6, ubiquitin, DNA binding and PCNA modification. Mol Carcinog 2007, 35(17):5819-5830.
• [56]Bailly V, Lamb J, Sung P, Prakash S, Prakash L: Specific complex formation between yeast RAD6 and RAD18 proteins: a potential mechanism for targeting RAD6 ubiquitin-conjugating activity to DNA damage sites. Genes Dev 1994, 8(7):811-820.
• [57]Jentsch S, McGrath J, Varshavsky A: The yeast DNA repair gene Rad6 encodes a ubiquitin-conjugating enzime. Nature 1987, 329(10):131-134.
• [58]Ulrich HD, Jentsch S: Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair. EMBO J 2000, 19(13):3388-3397.
• [59]Bailly V, Prakash S, Prakash L: Domains required for dimerization of yeast Rad6 ubiquitin-conjugating enzyme and Rad18 DNA binding protein. Mol Cell Biol 1997, 17(8):4536-4543.
• [60]Hibbert RG, Huang A, Boelens R, Sixma TK: E3 ligase Rad18 promotes monoubiquitination rather than ubiquitin chain formation by E2 enzyme Rad6. P Natl Acad Sci USA 2011, 1:1-7.
• [61]Worthylake D, Prakash S, Prakash L, Hill C: Crystal structure of the Saccharomyces cerevisiae ubiquitin-conjugating enzyme Rad6 at 2.6Å resolution. J Biol Chem 1998, 273(11):6271-6276.
• [62]Ulrich HD: Protein-protein interactions within an E2-RING finger complex. J Biol Chem 2003, 278(9):7051-7058.
• [63]Hofmann R, Pickart C: In vitro assembly and recognition of Lys-63 polyubiquitin chains. J Biol Chem 2001, 276(30):27936-43.
• [64]Eddins MJ, Carlile CM, Gomez KM, Pickart CM, Wolberger C: Mms2-Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation. Nat Struct Mol Biol 2006, 13(10):915-920.
• [65]Resnick M, Setlow J: Repair of pyrimidine dimer damage induced in yeast by ultraviolet light. J Bacteriol 1972, 109(3):979-986.
• [66]Fäth W, Brendel M: UV-induction of thymine-containing dimers in Saccharomyces cerevisiae. Z Naturforsch C 1975, 30(6):811-817.
• [67]Besozzi D, Cazzaniga P, Mauri G, Pescini D: BioSimWare: a software for the modeling, simulation and analysis of biological systems. In Membrane Computing, 11th International Conference, CMC 2010, Jena, Germany, August 24-27, Revised selected papers. Edited by Gheorghe M, Hinze T, Păun G, Rozenberg G, Salomaa A. LNCS 6501. Berlin, Heidelberg: Springer-Verlag; 2010:119-143.
• [68]Cao Y, Gillespie DT, Petzold L: Efficient step size selection for the tau-leaping simulation method. J Chem Phys 2006, 124(4):044109.
• [69]Morris M: Factorial sampling plans for preliminary computational experiments. Technometrics 1991, 33(2):161-174.
• [70]Campolongo F, Cariboni J, Saltelli A: An effective screening design for sensitivity analysis of large models. Environ Modell Softw 2007, 22(10):1509-1518.
• [71]Grabbe C, Husnjak K, Dikic I: The spatial and temporal organization of ubiquitin networks. Nat Rev Mol Cell Biol 2011, 12(5):295-307.
• [72]Weissman A: Themes and variations on ubiquitylation. Nat Rev Mol Cell Biol 2001, 2(3):169-178.
• [73]Eletr ZM, Huang DT, Duda DM, Schulman BA, Kuhlman B: E2 conjugating enzymes must disengage from their E1 enzymes before E3-dependent ubiquitin and ubiquitin-like transfer. Nat Struct Mol Biol 2005, 12(10):933-934.
• [74]Huang L, Kinnucan E, Wang G, Beaudenon S, Howley PM, Huibregtse JM, Pavletich NP: Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade. Science 1999, 286(5443):1321-6.
• [75]Zheng N, Wang P, Jeffrey PD, Pavletich NP: Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases. Cell 2000, 102(4):533-539.
• [76]Huang DT, Paydar A, Zhuang M, Waddell MB, Holton JM, Schulman BA: Structural basis for recruitment of Ubc12 by an E2 binding domain in NEDD8’s E1. Mol Cell 2005, 17(3):341-350.
• [77]Reverter D, Lima CD: Insights into E3 ligase activity revealed by a SUMO-RanGAP1-Ubc9-Nup358 complex. Nature 2005, 435(7042):687-692.
• [78]Zhang M, Windheim M, Roe SM, Peggie M, Cohen P, Prodromou C, Pearl LH: Chaperoned ubiquitylation–crystal structures of the CHIP U box E3 ubiquitin ligase and a CHIP-Ubc13-Uev1a complex. Mol Cell 2005, 20(4):525-538.
• [79]Bergink S, Jentsch S: Principles of ubiquitin and SUMO modifications in DNA repair. Nature 2009, 458:461-467.
• [80]Guex N, Peitsch M: SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 1997, 18(15):2714-2723.
• [81]Lee I, Schindelin H: Structural insights into E1-catalyzed ubiquitin activation and transfer to conjugating enzymes. Cell 2008, 134(2):268-278.
• [82]Li C, Donizelli M, Rodriguez N, Dharuri H, Endler L, Chelliah V, Li L, He E, Henry A, Stefan MI, Snoep JL, Hucka M, Le Novère N, Laibe C: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. BMC Syst Biol 2010, 4:92. BioMed Central Full Text
• [83]BioModels Database 2012. [http://www.ebi.ac.uk/biomodels-main webcite]
• [84]Hishida T, Kubota Y, Iwasaki H, AMCarr: RAD6–RAD18–RAD5–pathway-dependent tolerance to chronic low-dose ultraviolet light. Nature 2009, 457(7229):612-615.
• [85]Abdulovic A, Jinks-Robertson S: The in vivo characterization of translesion synthesis across UV-induced lesions in Saccharomyces cerevisiae: insights into Pol zeta- and Pol eta-dependent frameshift mutagenesis. Genetics 2006, 172(3):1489-1498.
• [86]Prakash L, Prakash S: Three additional genes involved in pyrimidine dimer removal in Saccharomyces cerevisiae: RAD7, RAD14 and MMS19. Mol Gen Genet 1979, 176(3):351-359.
• [87]Teng Y, Bennett M, Evans KE, Zhuang-Jackson H, Higgs A, Reed SH, Waters R: A novel method for the genome-wide high resolution analysis of DNA damage. Nucleic Acids Res 2011, 39(2):e10.
• [88]Lis E, Romesberg F: Role of Doa1 in the Saccharomyces cerevisiae DNA Damage Response. Mol Cell Biol 2006, 26(11):4122-4133.
• [89]Swaminathan S, Amerik A, Hochstrasser M: The Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast. Mol Cell Biol 1999, 10(8):2583-2594.
• [90]Amerik A, Hochstrasser M: Analysis of the deubiquitinating enzymes of the yeast Saccharomyces cerevisiae. Biol Chem 2000, 381(9-10):981-992.
• [91]Giannattasio M, Follonier C, Tourrière H, Puddu F, Lazzaro F, Pasero P, Lopes M, Plevani P, Muzi-Falconi M: Exo1 competes with repair synthesis, converts NER intermediates to long ssDNA gaps, and promotes checkpoint activation. Mol Cell 2010, 40:50-62.
• [92]Aboussekhra A, Al-Sharif I: Homologous recombination is involved in transcription-coupled repair of UV damage in Saccharomyces cerevisiae. EMBO J 2005, 24(11):1999-2010.
• [93]Santa Maria S, Gangavarapu V, Johnson R, Prakash L, Prakash S: Requirement of Nse1, a subunit of the Smc5-Smc6 complex, for Rad52-dependent postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae. Mol Cell Biol 2007, 27(23):8409-8418.
• [94]Gangavarapu V, Santa Maria S, Prakash S, Prakash L: Requirement of replication checkpoint protein kinases Mec1/Rad53 for postreplication repair in yeast. mBio 2011, 2(3):e00079—11.
• [95]Das-Bradoo S, Nguyen H, Wood J, Ricke R, Haworth J, Bielinsky A: Defects in DNA ligase I trigger PCNA ubiquitylation at Lys 107. Nat Cell Biol 2010, 12:74-79.
• [96]Holmes RM: Deubiquitinating enzymes and post-replication repair in Schizosaccharomyces pombe. PhD thesis, University of Sussex 2010
• [97]Gallego-Sánchez A, Andrés S, Conde F, San-Segundo P, Bueno A: Reversal of PCNA ubiquitylation by Ubp10 in Saccharomyces cerevisiae. PLoS Genet 2012, 8(7):e1002826.
• [98]Parker J, Bucceri A, Davies A, Heidrich K, Windecker H, Ulrich HD: SUMO modification of PCNA is controlled by DNA. EMBO J 2008, 27:2422-2431.