Abstract
DNA repair is a complex process that prevents genomic instability. Many proteins play fundamental roles in regulating the optimal repair of DNA lesions. Proliferating cell nuclear antigen (PCNA) is a key factor that initiates recombination-associated DNA synthesis after injury. Here, in very early S-phase, we show that the fluorescence intensity of mCherry-tagged PCNA after local micro-irradiation was less than the fluorescence intensity of non-irradiated mCherry-PCNA-positive replication foci. However, PCNA protein accumulated at locally irradiated chromatin in very late S-phase of the cell cycle, and this effect was more pronounced in the following G2 phase. In comparison to the dispersed form of PCNA, a reduced mobile fraction appeared in PCNA-positive replication foci during S-phase, and we observed similar recovery time after photobleaching at locally induced DNA lesions. This diffusion of mCherry-PCNA in micro-irradiated regions was not affected by cell cycle phases. We also studied the link between function of PCNA and A-type lamins in late S-phase. We found that the accumulation of PCNA at micro-irradiated chromatin is identical in wild-type and A-type lamin-deficient cells. Only micro-irradiation of the nuclear interior, and thus the irradiation of internal A-type lamins, caused the fluorescence intensity of mCherry-tagged PCNA to increase. In summary, we showed that PCNA begins to play a role in DNA repair in late S-phase and that PCNA function in repair is maintained during the G2 phase of the cell cycle. However, PCNA mobility is reduced after local micro-irradiation regardless of the cell cycle phase.
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References
Bártová E, Foltánková V, Legartová S, Sehnalová P, Sorokin DV, Suchánková J, Kozubek S (2014) Coilin is rapidly recruited to UVA-induced DNA lesions and γ-radiation affects localized movement of Cajal bodies. Nucleus 5:460–468. doi:10.4161/nucl.29229
Bravo R, Macdonald-Bravo H (1987) Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. J Cell Biol 105:1549–1554
Budhavarapu VN, Chavez M, Tyler JK (2013) How is epigenetic information maintained through DNA replication? Epigenetics. Chromatin 6:32. doi:10.1186/1756-8935-6-32
Cazzalini O, Sommatis S, Tillhon M, Dutto I, Bachi A, Rapp A, Nardo T, Scovassi AI, Necchi D, Cardoso MC, Stivala LA, Prosperi E (2004) CBP and p300 acetylate PCNA to link its degradation with nucleotide excision repair synthesis. Nucleic Acids Res 42:8433–8448. doi:10.1093/nar/gku533
Celis JE, Madsen P (1986) Increased nuclear cyclin/PCNA antigen staining of non S-phase transformed human amnion cells engaged in nucleotide excision DNA repair. FEBS Lett 209:277–283
Essers J, Theil AF, Baldeyron C, van Cappellen WA, Houtsmuller AB, Kanaar R, Vermeulen W (2005) Nuclear dynamics of PCNA in DNA replication and repair. Mol Cell Biol 25:9350–9359. doi:10.1128/MCB.25.21.9350-9359.2005
Hoogervorst EM, van Steeg H, de Vries A (2005) Nucleotide excision repair- and p53-deficient mouse models in cancer research. Mutat Res 574:3–21
Kelman Z (1997) PCNA: structure, functions and interactions. Oncogene 14:629–640. doi:10.1038/sj.onc.1200886
Li X, Stith CM, Burgers PM, Heyer WD (2009) PCNA is required for initiation of recombination-associated DNA synthesis by DNA polymerase delta. Mol Cell 36:704–713. doi:10.1016/j.molcel.2009.09.036
Maga G, Hubscher U (2003) Proliferating cell nuclear antigen (PCNA): a dancer with many partners. J Cell Sci 116:3051–3060. doi:10.1242/jcs.00653
Majka J, Burgers PM (2004) The PCNA-RFC families of DNA clamps and clamp loaders. Prog Nucleic Acid Res Mol Biol 78:227–260. doi:10.1016/S0079-6603(04)78006-X
Matsumoto Y, Kim K, Bogenhagen DF (1994) Proliferating cell nuclear antigen-dependent abasic site repair in Xenopus laevis oocytes: an alternative pathway of base excision DNA repair. Mol Cell Biol 14:6187–6197
McIlwraith MJ, Vaisman A, Liu Y, Fanning E, Woodgate R, West SC (2005) Human DNA polymerase eta promotes DNA synthesis from strand invasion intermediates of homologous recombination. Mol Cell 20:783–792. doi:10.1016/j.molcel.2005.10.001
Moldovan GL, Pfander B, Jentsch S (2007) PCNA, the maestro of the replication fork. Cell 129:665–679. doi:10.1016/j.cell.2007.05.003
Moldovan GL, Dejsuphong D, Petalcorin MI, Hofmann K, Takeda S, Boulton SJ, D'Andrea AD (2012) Inhibition of homologous recombination by the PCNA-interacting protein PARI. Mol Cell 45:75–86. doi:10.1016/j.molcel.2011.11.010
Naetar N, Hutter S, Dorner D, Dechat T, Korbei B, Gotzmann J, Beug H, Foisner R (2007) LAP2alpha-binding protein LINT-25 is a novel chromatin-associated protein involved in cell cycle exit. J Cell Sci 120:737–747. doi:10.1242/jcs.03390
Saleh-Gohari N, Helleday T (2004) Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells. Nucleic Acids Res 32:3683–3688. doi:10.1093/nar/gkh703
Sehnalová P, Legartová S, Cmarko D, Kozubek S, Bártová E (2014) Recruitment of HP1β to UVA-induced DNA lesions is independent of radiation-induced changes in A-type lamins. Biol Cell 106:151–165. doi:10.1111/boc.201300076
Shumaker DK, Solimando L, Sengupta K, Shimi T, Adam SA, Grunwald A, Strelkov SV, Aebi U, Cardoso MC, Goldman RD (2008) The highly conserved nuclear lamin Ig-fold binds to PCNA: its role in DNA replication. J Cell Biol 181:269–280. doi:10.1083/jcb.200708155
Smirnov E, Cmarko D, Kováčik L, Hagen GM, Popov A, Raska O, Prieto JL, Ryabchenko B, Amim F, McStay B, Raska I (2011) Replication timing of pseudo-NORs. J Struct Biol 173:213–218. doi:10.1016/j.jsb.2010.11.023
Smirnov E, Borkovec J, Kováčik L, Svidenská S, Schröfel A, Skalníková M, Švindrych Z, Křížek P, Ovesný M, Hagen GM, Juda P, Michalová K, Cardoso MC, Cmarko D, Raška I (2014) Separation of replication and transcription domains in nucleoli. J Struct Biol 188:259–266. doi:10.1016/j.jsb.2014.10.001
Smirnov E, Hornáček M, Kováčik L, Mazel T, Schröfel A, Svidenská S, Skalníková M, Bartová E, Cmarko D, Raška I (2016) Reproduction of the FC/DFC units in nucleoli. Nucleus 7:203–215. doi:10.1080/19491034.2016.1157674
Solomon DA, Cardoso MC, Knudsen ES (2004) Dynamic targeting of the replication machinery to sites of DNA damage. J Cell Biol 166:455–463. doi:10.1083/jcb.200312048
Soutoglou E, Misteli T (2008) Activation of the cellular DNA damage response in the absence of DNA lesions. Science 320:1507–1510. doi:10.1126/science.1159051
Strzalka W, Ziemienowicz A (2011) Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation. Ann Bot 107:1127–1140. doi:10.1093/aob/mcq243
Suchankova J, Kozubek S, Legartova S, Sehnalova P, Kuntziger T, Bartova E (2015) Distinct kinetics of DNA repair protein accumulation at DNA lesions and cell cycle-dependent formation of gammaH2AX- and NBS1-positive repair foci. Biol Cell 107:440–454. doi:10.1111/boc.201500050
Sullivan T, Escalante-Alcalde D, Bhatt H, Anver M, Bhat N, Nagashima K, Stewart CL, Burke B (1999) Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy. J Cell Biol 147:913–920
Toschi L, Bravo R (1988) Changes in cyclin/proliferating cell nuclear antigen distribution during DNA repair synthesis. J Cell Biol 107:1623–1628
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This work was supported by Czech Science Foundation grant number P302–12-G157. The research leading to these results received funding from the Norwegian Financial Mechanism 2009–2014 under project contract no. 7F14369.
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Handling editor: Pavel Dráber
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Supplementary Figure 1
Accumulation of mCherry-tagged PCNA at locally micro-irradiated chromatin containing DNA lesions. A 405-nm laser diode was used for local micro-irradiation. Based on the PCNA nuclear distribution pattern, analyses were performed in (Aa) non-S-phase, (Ab) early S-phase, and (Ac) late S-phase in HeLa cells stably expressing GFP-tagged histone H2B (green) and transiently expressing mCherry-PCNA (red). (GIF 262 kb)
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Bártová, E., Suchánková, J., Legartová, S. et al. PCNA is recruited to irradiated chromatin in late S-phase and is most pronounced in G2 phase of the cell cycle. Protoplasma 254, 2035–2043 (2017). https://doi.org/10.1007/s00709-017-1076-1
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DOI: https://doi.org/10.1007/s00709-017-1076-1