Abstract
The Ku70 protein is involved in numerous cell functions, the nonhomologous end joining (NHEJ) DNA repair pathway being the best known. Here, we report a novel function for this protein in the grasshopper Eyprepocnemis plorans. We observed the presence of large Ku70 foci on the centromeres of meiotic and mitotic chromosomes during the cell cycle stages showing the highest centromeric activity (i.e., metaphase and anaphase). The fact that colchicine treatment prevented centromeric location of Ku70, suggests a microtubule-dependent centromeric function for Ku70. Likewise, the absence of Ku70 at metaphase–anaphase centromeres from three males whose Ku70 gene had been knocked down using interference RNA, and the dramatic increase in the frequency of polyploid spermatids observed in these males, suggest that the centromeric presence of Ku70 is required for normal cytokinesis in this species. The centromeric function of Ku70 was not observed in 14 other grasshopper and locust species, or in the mouse, thus suggesting that it is an autapomorphy in E. plorans.
Similar content being viewed by others
Abbreviations
- +TIPs:
-
Plus ends of growing microtubules
- APC:
-
Adenomatous polyposis coli protein
- DAPI:
-
4′,6-diamidino-2-phenylindole
- Dffi:
-
Days from the first injection
- DNA-PKcs:
-
DNA-dependent protein kinase catalytic subunit
- DSB:
-
Double-stranded DNA breaks
- EB1:
-
End binding 1 protein
- FIAD:
-
Feulgen image analysis densitometry
- γH2AX:
-
Phosphorylated form of the H2AX histone
- GAPDH:
-
Glyceraldehyde-3-phosphate dehydrogenase
- HR:
-
Homologous recombination
- NHEJ:
-
Nonhomologous end joining
- RNAi:
-
Interference RNA
- RP49:
-
Ribosomal protein 49
- SAC:
-
Spindle assembly checkpoint
References
Åström SU, Okamura SM, Rine J (1999) Yeast cell-type regulation of DNA repair. Nature 397:310
Bertinato J, Schild-Poulter C, Hache RJG (2001) Nuclear localization of Ku antigen is promoted independently by basic motifs in the Ku70 and Ku80 subunits. J Cell Sci 114:89–99
Bhattacharyya B, Pnada D, Gupta S, Banerjee M (2008) Anti-mitotic activity of colchicine and the structural basis for its interaction with tubulin. Med Res Rev 28:155–183
Cabrero J, Teruel M, Carmona FD, Camacho JPM (2007a) Histone H2AX phosphorylation is associated with most meiotic events in grasshopper. Cytogenet Genome Res 116:311–315
Cabrero J, Palomino-Morales RJ, Camacho JPM (2007b) The DNA-repair Ku70 protein is located in the nucleus and tail of elongating spermatids in grasshoppers. Chromosome Res 15:1093–1100
Calvente A, Viera A, Page J et al (2005) DNA-double strand breaks and homology search: inferences from a species with incomplete pairing and synapsis. J Cell Sci 118:2957–2963
Camacho JPM, Carballo AR, Cabrero J (1980) The B-chromosome system of the grasshopper Eyprepocnemis plorans subsp. plorans (Charpentier). Chromosoma 80:163–176
Camacho JPM, Cabrero J, Viseras E, López-León MD, Navas-Castillo J, Alché JD (1991) G-banding in two species of grasshoppers and its relationship to C, N and fluorescence banding techniques. Genome 34:638–643
Camacho JPM, Bakkali M, Corral JM et al (2002) Host recombination is dependent on the degree of parasitism. Proc R Soc B 269:2173–2177
Dikovskaya D, Schiffmann D, Newton IP et al (2007) Loss of APC induces polyploidy as a result of a combination of defects in mitosis and apoptosis. J Cell Biol 176:183–195
Dong Y, Friedrich M (2005) Nymphal RNAi: systemic RNAi mediated gene knockdown in juvenile grasshopper. BMC Biotechnol 5:25
Fox DP, Hewitt GM, Hall DJ (1974) DNA replication and RNA transcription of euchromatic and heterochromatic chromosome regions during grasshopper meiosis. Chromosoma 45:43–62
Gao YJ, Chaudhuri J, Zhu CM, Davidson L, Weaver DT, Alt FW (1998) A targeted DNA-PKcs-null mutation reveals DNA-PK-independent functions for KU in V(D)J recombination. Immunity 9:367–376
Gell D, Jackson SP (1999) Mapping of protein-protein interactions within the DNA-dependent protein kinase complex. Nucl Acids Res 27:3494–3502
Goedecke W, Eijpe M, Offenberg HH, van Aalderen M, Heyting C (1999) Mre11 and Ku70 interact in somatic cells, but are differentially expressed in early meiosis. Nat Genet 23:194–198
Green RA, Wollman R, Kaplan KB (2005) APC and EB1 function together in mitosis to regulate spindle dynamics and chromosome alignment. Mol Biol Cell 16:4609–4622
Gu YS, Seidl KJ, Rathbun GA et al (1997) Growth retardation and leaky SCID phenotype of Ku70-deficient mice. Immunity 7:653–665
Gu YS, Sekiguchi J, Gao YJ et al (2000) Defective embryonic neurogenesis in Ku-deficient but not DNA-dependent protein kinase catalytic subunit-deficient mice. Proc Natl Acad Sci USA 97:2668–2673
Howell BJ, Moree B, Farrar EM, Stewart S, Fang G, Salmon ED (2004) Spindle checkpoint protein dynamics at kinetochores in living cells. Curr Biol 14:953–964
Kang SW, Shin YJ, Shim YJ, Jeong SY, Park IS, Min BH (2005) Clusterin interacts with SCLIP (SCG10-like protein) and promotes neurite outgrowth of PC12 cells. Exp Cell Res 309:305–315
Kaplan KB, Burds AA, Swedlow JR, Bekir SS, Sorger PK, Näthke IS (2001) A role for the adenomatous polyposis coli protein in chromosome segregation. Nat Cell Biol 3:429–432
Katz DJ, Beer MA, Levorse JM, Tilghman SM (2005) Functional characterization of a novel Ku70/80 pause site at the H19/Igf2 imprinting control region. Mol Cell Biol 25:3855–3863
Koike M (2002) Dimerization, translocation and localization of Ku70 and Ku80 proteins. J Rad Res 43:223–236
Koike M, Koike A (2005) The Ku70-binding site of Ku80 is required for the stabilization of Ku70 in the cytoplasm, for the nuclear translocation of Ku80, and for Ku80-dependent DNA repair. Exp Cell Res 305:266–276
Koike M, Awaji T, Kataoka M et al (1999) Differential subcellular localization of DNA-dependent protein kinase components Ku and DNA-PKcs during mitosis. J Cell Sci 112:4031–4039
Koike M, Shiomi T, Koike A (2001) Dimerization and nuclear localization of Ku proteins. J Biol Chem 276:11167–11173
Lee SE, Pâques F, Sylvan J, Haber JE (1999) Role of yeast SIR genes and mating type in directing DNA double-strand breaks to homologous and non-homologous repair paths. Curr Biol 9:767–770
Lee K-J, Lin Y-F, Chou H-Y et al (2011) Involvement of DNA-dependent protein kinase in normal cell cycle progression through mitosis. J Biol Chem 286:12796–12802
Martinez JJ, Seveau S, Veiga E, Matsuyama S, Cossart P (2005) Ku70, a component of DNA-dependent protein kinase, is a mammalian receptor for Rickettsia conorii. Cell 123:1013–1023
Monferran S, Muller C, Mourey L, Frit P, Salles B (2004a) The membrane-associated form of the DNA repair protein Ku is involved in cell adhesion to fibronectin. J Mol Biol 337:503–511
Monferran S, Paupert J, Dauvillier S, Salles B, Muller C (2004b) The membrane form of the DNA repair protein Ku interacts at the cell surface with metalloproteinase 9. EMBO J 23:3758–3768
Muñoz E, Perfectti F, Martín-Alganza A, Camacho JPM (1998) Parallel effects of a B chromosome and a mite that decrease female fitness in the grasshopper Eyprepocnemis plorans. Proc Roy Soc B 265:1903–1909
Normand G, King RW (2010) Understanding cytokinesis failure. In: Poon, R.Y.C. (ed) Polyploidization and cancer. Springer, New York
Nussenzweig A, Chen C, da Costa Soares V, Sanchez M, Sokol K, Nussenzweig MC, Li GC (1996) Requirement for Ku80 in growth and immunoglobulin V(D)J recombination. Nature 382:551–555
Paull TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M, Bonner WM (2000) A critical role for histone H2AX in sequential recruitment of repair factors to nuclear foci after DNA damage. Curr Biol 10:886–895
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res 29:2002–2007
Pöggeler S, Kuck U (2006) Highly efficient generation of signal transduction knockout mutants using a fungal strain deficient in the mammalian ku70 ortholog. Gene 378:1–10
Rieder CL, Palazzo RE (1992) Colcemid and the mitotic cycle. J Cell Sci 102:387–392
Rodgers W, Jordan SJ, Capra JD (2002) Transient association of Ku with nuclear substrates characterized using fluorescence photobleaching. J Immunol 168:2348–2355
Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana Press, Totowa, pp 365–386
Ruiz-Ruano FJ, Ruiz-Estévez M, Rodríguez-Pérez J, López-Pino JL, Cabrero J, Camacho JPM (2011) DNA amount of X and B chromosomes in the grasshoppers Eyprepocnemis plorans and Locusta migratoria. Cytogenet Genome Res 134:120–126
Sawada M, Sun WY, Hayes P, Leskov K, Boothman DA, Matsuyama S (2003) Ku70 suppresses the apoptotic translocation of Bax to mitochondria. Nat Cell Biol 5:320–329
Scherthan H, Trelles-Sticken E (2008) Absence of yKu/Hdf1 but not myosin-like proteins alters chromosome dynamics during prophase I in yeast. Differentiation 76:91–98
Schweitzer JK, D’Souza-Schorey C (2005) A requirement for ARF6 during the completion of cytokinesis. Exp Cell Res 311:74–83
Shi Q, King RW (2005) Chromosome nondisjunction yields tetraploid rather than aneuploid cells in human cell lines. Nature 437:1038–1042
Shuaib M, Ouararhni K, Dimitrov S, Hamiche A (2010) HJURP binds CENP-A via a highly conserved N-terminal domain and mediates its deposition at centromeres. Proc Natl Acad Sci USA 107:1349–1354
Singson A (2001) Every sperm is sacred: fertilization in Caenorhabditis elegans. Dev Biol 230:101–109
Song KY, Jung YS, Jung DH, Lee I (2001) Human Ku70 interacts with heterochromatin protein 1 alpha. J Biol Chem 276:8321–8327
Takata M, Sasaki MS, Sonoda E et al (1998) Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J 17:5497–5508
Tirnauer JS, Canman JC, Salmon ED, Mitchison TJ (2002) EB1 targets to kinetochores with attached, polymerizing microtubules. Mol Biol Cell 13:4308–4316
Tuteja R, Tuteja N (2000) Ku autoantigen: a multifunctional DNA-binding protein. Crit Rev Biochem Mol Biol 35:1–33
Van Hiel MB, Van Wielendaele P, Temmerman L et al (2009) Identification and validation of housekeeping genes in brains of the desert locust Schistocerca gregaria under different developmental conditions. BMC Mol Biol 10:56
Vandesompele J, De Preter K, Pattyn F et al (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:7
Viera A, Santos JL, Page J et al (2004) DNA double-strand-break, recombination and synapsis, the timing of meiosis differs in grasshoppers and flies. EMBO Rep 5:385–391
Yang CR, Yeh SY, Leskov K et al (1999) Isolation of Ku70-binding proteins (KUBs). Nucleic Acids Res 27:2165–2174
Zhou B, Elledge SJ (2000) The DNA damage response: putting checkpoints in perspective. Nature 408:433–439
Zurita S, Cabrero J, López-León MD, Camacho JPM (1998) Polymorphism regeneration for a neutralized selfish B chromosome. Evolution 52:274–277
Acknowledgments
We thank R. Jiménez for providing us with mouse spermatocytes, M. Ruiz-Estévez for giving us an E. plorans cDNA aliquote, and T. López for technical assistance. This study was supported by grants from the Spanish Ministerio de Ciencia e Innovación (CGL2009-11917 and BFU2010-16438) and Plan Andaluz de Investigación (CVI-6649), and was partially performed by FEDER funds. M. Bakkali was supported by a Ramón y Cajal fellowship from the Spanish Ministerio de Ciencia e Innovación.
Author information
Authors and Affiliations
Corresponding author
Additional information
Responsible Editor: Beth A. Sullivan.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Fig. S1
The centromeric location of Ku70 also occurs during mitosis in E. plorans. a, b Spermatogonial mitotic metaphase. c, d Embryo mitotic metaphase cell. e, f Embryo mitotic metaphase from a neuroblast cell (of giant size). Note the presence of centromeric foci of Ku70 in the three cells. This was observed in 100 % of the analyzed cells. Bar = 10 μm. (JPEG 94 kb)
Fig. S2
Absence of centromeric immunofluorescence signals for Ku70 at meiotic metaphase I in 14 grasshopper species and the mouse. Examples are shown for Ku70 (a, c, e) and DAPI+Ku70 (b, d, f) in the grasshopper species O. panteli (a, b) and S. azurescens (c, d), and the mouse (e, f). Bar = 10 μm. (JPEG 41 kb)
Fig. S3
Presence of polyploid and binucleate cells in RNAi males. a Two tetraploid spermatogonia at mitotic prometaphase. b Tetraploid metaphase I cell. c Binucleate cell. (JPEG 52 kb)
Rights and permissions
About this article
Cite this article
Cabrero, J., Bakkali, M., Navarro-Domínguez, B. et al. The Ku70 DNA-repair protein is involved in centromere function in a grasshopper species. Chromosome Res 21, 393–406 (2013). https://doi.org/10.1007/s10577-013-9367-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10577-013-9367-7