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H2AX foci in late S/G2- and M-phase cells after hydroxyurea- and aphidicolin-induced DNA replication stress in Vicia

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Abstract

Immunocytochemistry using α-phospho-H2AX antibodies shows that hydroxyurea (HU), an inhibitor of ribonucleotide reductase, and aphidicolin (APH), an inhibitor of DNA-polymerases α and δ, may promote formation of phospho-H2AX foci in late S/G2-phase cells in root meristems of Vicia faba. Although fluorescent foci spread throughout the whole area of nucleoplasm, large phospho-H2AX aggregates in HU-treated cells allocate mainly in perinucleolar regions. A strong tendency of ATR/ATM-dependent phospho-Chk1S317 kinase to focus in analogous compartments, as opposed to phospho-Chk2T68 and to both effector kinases in APH-treated cells, may suggest that selected elements of the intra-S-phase cell cycle checkpoints share overlapping locations with DNA repair factors known to concentrate in phospho-H2AX aggregates. APH-induced phosphorylation of H2AX exhibits little or no overlap with the areas positioned close to nucleoli. Following G2-M transition of the HU- and APH-pretreated cells, altered chromatin structures are still discernible as large phospho-H2AX foci in the vicinity of chromosomes. Both in HU- and APH-treated roots, immunofluorescence analysis revealed a dominant fraction of small foci and a less frequent population of large phospho-H2AX agregates, similar to those observed in animal cells exposed to ionizing radiation. The extent of H2AX phosphorylation has been found considerably reduced in root meristem cells treated with HU and caffeine. The frequencies of phospho-H2AX foci observed during mitosis and caffeine-mediated premature chromosome condensation (PCC) suggest that there may be functional links between the checkpoint mechanisms that control genome integrity and those activities which operate throughout the unperturbed mitosis in plants.

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Abbreviations

APH:

Aphidicolin

DSB:

Double strand breaks

HU:

Hydroxyurea

PCC:

Premature chromosome condensation

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Acknowledgments

This work was supported by the National Committee of Scientific Research, grant 2PO4C 044 27.

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Authors and Affiliations

  1. Department of Cytophysiology, University of Łódź, ul. Pilarskiego 14, 90231, Łódź, Poland

    Dorota Rybaczek, Aleksandra Bodys & Janusz Maszewski

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  1. Dorota Rybaczek
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  2. Aleksandra Bodys
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  3. Janusz Maszewski
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Correspondence to Dorota Rybaczek.

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Rybaczek, D., Bodys, A. & Maszewski, J. H2AX foci in late S/G2- and M-phase cells after hydroxyurea- and aphidicolin-induced DNA replication stress in Vicia . Histochem Cell Biol 128, 227–241 (2007). https://doi.org/10.1007/s00418-007-0311-9

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