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The Ku70 DNA-repair protein is involved in centromere function in a grasshopper species

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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.

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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

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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.

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Authors and Affiliations

  1. Departamento de Genética, Facultad de Ciencias, Universidad de Granada, Granada, Spain

    Josefa Cabrero, Mohammed Bakkali, Beatriz Navarro-Domínguez, Francisco J. Ruíz-Ruano, Rubén Martín-Blázquez, María Dolores López-León & Juan Pedro M. Camacho

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  1. Josefa Cabrero
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  2. Mohammed Bakkali
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  3. Beatriz Navarro-Domínguez
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  4. Francisco J. Ruíz-Ruano
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  5. Rubén Martín-Blázquez
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  6. María Dolores López-León
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  7. Juan Pedro M. Camacho
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Correspondence to Juan Pedro M. Camacho.

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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)

High-resolution image (TIFF 3912 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)

High-resolution image (TIFF 1780 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)

High-resolution image (TIFF 3201 kb)

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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

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