Deficiency in chromatin assembly factor-1 (CAF-1) in plants through dysfunction of its components, FASCIATA1 and 2 (FAS1, FAS2), leads to the specific and progressive loss of rDNA and telomere repeats in plants. This loss is attributed to defective repair mechanisms for the increased DNA breaks encountered during replication, a consequence of impaired replication-dependent chromatin assembly. In this study, we explore the role of KU70 in these processes. Our findings reveal that, although the rDNA copy number is reduced in ku70 mutants when compared with wild-type plants, it is not markedly affected by diverse KU70 status in fas1 mutants. This is consistent with our previous characterisation of rDNA loss in fas mutants as a consequence part of the single-strand annealing pathway of homology-dependent repair. In stark contrast to rDNA, KU70 dysfunction fully suppresses the loss of telomeres in fas1 plants and converts telomeres to their elongated and heterogeneous state typical for ku70 plants. We conclude that the alternative telomere lengthening pathway, known to be activated in the absence of KU70, overrides progressive telomere loss due to CAF-1 dysfunction.

Biology Centre Czech Academy of Sciences Institute of Plant Molecular Biology České Budějovice Czech Republic

Department of Cell Biology and Radiobiology Institute of Biophysics of the Czech Academy of Sciences v v i Brno CZ 61265 Czech Republic

Laboratory of Functional Genomics and Proteomics NCBR Faculty of Science Masaryk University Brno CZ 61137 Czech Republic

Mendel Centre for Plant Genomics and Proteomics CEITEC Masaryk University Brno CZ 62500 Czech Republic

University of South Bohemia Faculty of Science České Budějovice Czech Republic

Zobrazit více v PubMed

Adamusova, K., Khosravi, S., Fujimoto, S., Houben, A., Matsunaga, S., Fajkus, J. et al. (2020) Two combinatorial patterns of telomere histone marks in plants with canonical and non‐canonical telomere repeats. The Plant Journal, 102, 678–687.

Benoit, M., Simon, L., Desset, S., Duc, C., Cotterell, S., Poulet, A. et al. (2019) Replication‐coupled histone H3.1 deposition determines nucleosome composition and heterochromatin dynamics during Arabidopsis seedling development. The New Phytologist, 221, 385–398.

Bryan, T.M., Marusic, L., Bacchetti, S., Namba, M. & Reddel, R.R. (1997) The telomere lengthening mechanism in telomerase‐negative immortal human cells does not involve the telomerase RNA subunit. Human Molecular Genetics, 6, 921–926.

Bryan, T.M. & Reddel, R.R. (1997) Telomere dynamics and telomerase activity in in vitro immortalised human cells. European Journal of Cancer, 33, 767–773.

Bundock, P., van Attikum, H. & Hooykaas, P. (2002) Increased telomere length and hypersensitivity to DNA damaging agents in an Arabidopsis KU70 mutant. Nucleic Acids Research, 30, 3395–3400.

Citovsky, V., Lee, L.Y., Vyas, S., Glick, E., Chen, M.H., Vainstein, A. et al. (2006) Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta. Journal of Molecular Biology, 362, 1120–1131.

Culligan, K., Tissier, A. & Britt, A. (2004) ATR regulates a G2‐phase cell‐cycle checkpoint in Arabidopsis thaliana. Plant Cell, 16, 1091–1104.

Dellaporta, S.L., Wood, J. & Hicks, J.B. (1983) A plant DNA minipreparation: Version II. Plant Mol. Biol. Rep., 1, 19–21.

Dvorackova, M., Fojtova, M. & Fajkus, J. (2015) Chromatin dynamics of plant telomeres and ribosomal genes. The Plant Journal, 83, 18–37.

Eekhout, T., Dvorackova, M., Pedroza Garcia, J.A., Nespor Dadejova, M., Kalhorzadeh, P., Van den Daele, H. et al. (2021) G2/M‐checkpoint activation in fasciata1 rescues an aberrant S‐phase checkpoint but causes genome instability. Plant Physiology, 186, 1893–1907.

Fojtová, M., Fajkus, P., Polanská, P. & Fajkus, J. (2015) Terminal Restriction Fragments (TRF) Method to Analyze Telomere Lengths. Bio-protocol, 5(23), e1671.

Gallego, M.E., Bleuyard, J.Y., Daoudal‐Cotterell, S., Jallut, N. & White, C.I. (2003) Ku80 plays a role in non‐homologous recombination but is not required for T‐DNA integration in Arabidopsis. The Plant Journal: For Cell and Molecular Biology, 35, 557–565.

Gallego, M.E., Jalut, N. & White, C.I. (2003) Telomerase dependence of telomere lengthening in Ku80 mutant Arabidopsis. Plant Cell, 15, 782–789.

Garcia, V., Bruchet, H., Camescasse, D., Granier, F., Bouchez, D. & Tissier, A. (2003) AtATM is essential for meiosis and the somatic response to DNA damage in plants. Plant Cell, 15, 119–132.

Griffith, J.D., Comeau, L., Rosenfield, S., Stansel, R.M., Bianchi, A., Moss, H. et al. (1999) Mammalian telomeres end in a large duplex loop. Cell, 97, 503–514.

Hisanaga, T., Ferjani, A., Horiguchi, G., Ishikawa, N., Fujikura, U., Kubo, M. et al. (2013) The ATM‐dependent DNA damage response acts as an upstream trigger for compensation in the fas1 mutation during Arabidopsis leaf development. Plant Physiology, 162, 831–841.

Hoek, M., Myers, M.P. & Stillman, B. (2011) An analysis of CAF‐1‐interacting proteins reveals dynamic and direct interactions with the KU complex and 14‐3‐3 proteins. The Journal of Biological Chemistry, 286, 10876–10887.

Ijdo, J.W., Wells, R.A., Baldini, A. & Reeders, S.T. (1991) Improved telomere detection using a telomere repeat probe (TTAGGG)n generated by PCR. Nucleic Acids Research, 19, 4780.

Jaske, K., Mokros, P., Mozgova, I., Fojtova, M. & Fajkus, J. (2013) A telomerase‐independent component of telomere loss in chromatin assembly factor 1 mutants of Arabidopsis thaliana. Chromosoma, 122, 285–293.

Kaya, H., Shibahara, K.I., Taoka, K.I., Iwabuchi, M., Stillman, B. & Araki, T. (2001) FASCIATA genes for chromatin assembly factor‐1 in arabidopsis maintain the cellular organization of apical meristems. Cell, 104, 131–142.

Kazda, A., Zellinger, B., Rossler, M., Derboven, E., Kusenda, B. & Riha, K. (2012) Chromosome end protection by blunt‐ended telomeres. Genes & Development, 26, 1703–1713.

Kirik, A., Pecinka, A., Wendeler, E. & Reiss, B. (2006) The chromatin assembly factor subunit FASCIATA1 is involved in homologous recombination in plants. Plant Cell, 18, 2431–2442.

Kolarova, K., Nespor Dadejova, M., Loja, T., Lochmanova, G., Sykorova, E. & Dvorackova, M. (2021) Disruption of NAP1 genes in Arabidopsis thaliana suppresses the fas1 mutant phenotype, enhances genome stability and changes chromatin compaction. The Plant Journal, 106, 56–73.

Li, B., Reddy, S. & Comai, L. (2011) Depletion of Ku70/80 reduces the levels of extrachromosomal telomeric circles and inhibits proliferation of ALT cells. Aging (Albany NY), 3, 395–406.

Lycka, M., Peska, V., Demko, M., Spyroglou, I., Kilar, A., Fajkus, J. et al. (2021) WALTER: an easy way to online evaluate telomere lengths from terminal restriction fragment analysis. BMC Bioinformatics, 22, 145.

Mozgova, I., Mokros, P. & Fajkus, J. (2010) Dysfunction of chromatin assembly factor 1 induces shortening of telomeres and loss of 45S rDNA in Arabidopsis thaliana. Plant Cell, 22, 2768–2780.

Mozgova, I., Wildhaber, T., Trejo‐Arellano, M.S., Fajkus, J., Roszak, P., Kohler, C. et al. (2018) Transgenerational phenotype aggravation in CAF‐1 mutants reveals parent‐of‐origin specific epigenetic inheritance. The New Phytologist, 220, 908–921.

Muchova, V., Amiard, S., Mozgova, I., Dvorackova, M., Gallego, M.E., White, C. et al. (2015) Homology‐dependent repair is involved in 45S rDNA loss in plant CAF‐1 mutants. The Plant Journal, 81, 198–209.

Murray, M.G. & Thompson, W.F. (1980) Rapid isolation of high molecular‐weight plant DNA. Nucleic Acids Research, 8, 4321–4325.

Otero, S., Desvoyes, B., Peiro, R. & Gutierrez, C. (2016) Histone H3 dynamics reveal domains with distinct proliferation potential in the Arabidopsis root. Plant Cell, 28, 1361–1371.

Pavlistova, V., Dvorackova, M., Jez, M., Mozgova, I., Mokros, P. & Fajkus, J. (2016) Phenotypic reversion in FAS mutants of Arabidopsis thaliana by reintroduction of FAS genes: variable recovery of telomeres with major spatial rearrangements and transcriptional reprogramming of 45S rDNA genes. The Plant Journal, 88, 411–424.

Picart‐Picolo, A., Grob, S., Picault, N., Franek, M., Llauro, C., Halter, T. et al. (2020) Large tandem duplications affect gene expression, 3D organization, and plant‐pathogen response. Genome Research, 30, 1583–1592.

Pontvianne, F., Blevins, T., Chandrasekhara, C., Mozgova, I., Hassel, C., Pontes, O.M. et al. (2013) Subnuclear partitioning of rRNA genes between the nucleolus and nucleoplasm reflects alternative epiallelic states. Genes & Development, 27, 1545–1550.

Ramirez‐Parra, E. & Gutierrez, C. (2007) E2F regulates FASCIATA1, a chromatin assembly gene whose loss switches on the endocycle and activates gene expression by changing the epigenetic status. Plant Physiology, 144, 105–120.

Riha, K., McKnight, T.D., Griffing, L.R. & Shippen, D.E. (2001) Living with genome instability: plant responses to telomere dysfunction. Science, 291, 1797–1800.

Riha, K., Watson, J.M., Parkey, J. & Shippen, D.E. (2002) Telomere length deregulation and enhanced sensitivity to genotoxic stress in Arabidopsis mutants deficient in Ku70. EMBO Journal, 21, 2819–2826.

Ruckova, E., Friml, J., Prochazkova Schrumpfova, P. & Fajkus, J. (2008) Role of alternative telomere lengthening unmasked in telomerase knock‐out mutant plants. Plant Molecular Biology, 66, 637–646.

Schonrock, N., Exner, V., Probst, A., Gruissem, W. & Hennig, L. (2006) Functional genomic analysis of CAF‐1 mutants in Arabidopsis thaliana. The Journal of Biological Chemistry, 281, 9560–9568.

Shakirov, E.V. & Shippen, D.E. (2004) Length regulation and dynamics of individual telomere tracts in wild‐type Arabidopsis. Plant Cell, 16, 1959–1967.

Sims, J., Copenhaver, G.P. & Schlogelhofer, P. (2019) Meiotic DNA repair in the nucleolus employs a nonhomologous end‐joining mechanism. Plant Cell, 31, 2259–2275.

Sovakova, P.P., Magdolenova, A., Konecna, K., Rajecka, V., Fajkus, J. & Fojtova, M. (2018) Telomere elongation upon transfer to callus culture reflects the reprogramming of telomere stability control in Arabidopsis. Plant Molecular Biology, 98, 81–99.

Sykorova, E., Leitch, A.R. & Fajkus, J. (2006) Asparagales telomerases which synthesize the human type of telomeres. Plant Molecular Biology, 60, 633–646.

Valuchova, S., Fulnecek, J., Prokop, Z., Stolt‐Bergner, P., Janouskova, E., Hofr, C. et al. (2017) Protection of Arabidopsis blunt‐ended telomeres is mediated by a physical association with the Ku heterodimer. Plant Cell, 29, 1533–1545.

Varas, J., Sanchez‐Moran, E., Copenhaver, G.P., Santos, J.L. & Pradillo, M. (2015) Analysis of the relationships between DNA double‐Strand breaks, synaptonemal complex and crossovers using the Atfas1‐4 mutant. PLoS Genetics, 11, e1005301.

Varas, J., Santos, J.L. & Pradillo, M. (2017) The absence of the Arabidopsis chaperone complex CAF‐1 produces mitotic chromosome abnormalities and changes in the expression profiles of genes involved in DNA repair. Frontiers in Plant Science, 8, 525.

Watson, J.M. & Shippen, D.E. (2007) Telomere rapid deletion regulates telomere length in Arabidopsis thaliana. Molecular and Cellular Biology, 27, 1706–1715.

Wu, W., He, J.N., Lan, M., Zhang, P. & Chu, W.K. (2021) Transcription‐replication collisions and chromosome fragility. Frontiers in Genetics, 12, 804547.

Yu, E.Y., Perez‐Martin, J., Holloman, W.K. & Lue, N.F. (2015) Mre11 and Blm‐dependent formation of ALT‐like telomeres in Ku‐deficient Ustilago maydis. PLoS Genetics, 11, e1005570.

Zachova, D., Fojtova, M., Dvorackova, M., Mozgova, I., Lermontova, I., Peska, V. et al. (2013) Structure‐function relationships during transgenic telomerase expression in Arabidopsis. Physiologia Plantarum, 149, 114–126.

Zellinger, B., Akimcheva, S., Puizina, J., Schirato, M. & Riha, K. (2007) Ku suppresses formation of telomeric circles and alternative telomere lengthening in Arabidopsis. Molecular Cell, 27, 163–169.

Zhou, W., Gao, J., Ma, J., Cao, L., Zhang, C., Zhu, Y. et al. (2016) Distinct roles of the histone chaperones NAP1 and NRP and the chromatin‐remodeling factor INO80 in somatic homologous recombination in Arabidopsis thaliana. The Plant Journal: For Cell and Molecular Biology, 88, 397–410.

Leishmania mexicana telomeres at high resolution: Ku80, TERT, and alternative lengthening mechanisms