title: DNA replication stress induces deregulation of the cell cycle events in root meristems of Allium cepa.
abstract: OBJECTIVE
  Prolonged treatment of Allium cepa root meristems with changing concentrations of hydroxyurea (HU) results in either premature chromosome condensation or cell nuclei with an uncommon form of biphasic chromatin organization. The aim of the current study was to assess conditions that compromise cell cycle checkpoints and convert DNA replication stress into an abnormal course of mitosis.
  
  
  METHODS
  Interphase-mitotic (IM) cells showing gradual changes of chromatin condensation were obtained following continuous 72 h treatment of seedlings with 0·75 mm HU (without renewal of the medium). HU-treated root meristems were analysed using histochemical stainings (DNA-DAPI/Feulgen; starch-iodide and DAB staining for H(2)O(2) production), Western blotting [cyclin B-like (CBL) proteins] and immunochemistry (BrdU incorporation, detection of γ-H2AX and H3S10 phosphorylation).
  
  
  RESULTS
  Continuous treatment of onion seedlings with a low concentration of HU results in shorter root meristems, enhanced production of H(2)O(2), γ-phosphorylation of H2AX histones and accumulation of CBL proteins. HU-induced replication stress gives rise to axially elongated cells with half interphase/half mitotic structures (IM-cells) having both decondensed and condensed domains of chromatin. Long-term HU treatment results in cell nuclei resuming S phase with gradients of BrdU labelling. This suggests a polarized distribution of factors needed to re-initiate stalled replication forks. Furthermore, prolonged HU treatment extends both the relative time span and the spatial scale of H3S10 phosphorylation known in plants.
  
  
  CONCLUSIONS
  The minimum cell length and a threshold level of accumulated CBL proteins are both determining factors by which the nucleus attains commitment to induce an asynchronous course of chromosome condensation. Replication stress-induced alterations in an orderly route of the cell cycle events probably reflect a considerable reprogramming of metabolic functions of chromatin combined with gradients of morphological changes spread along the nucleus.
authors.affiliation: Department of Cytophysiology, Faculty of Biology and Environmental Protection, University of Łódź, Poland. zabkka@poczta.onet.pl
authors.forename: Aneta
authors.initials: A
authors.lastname: Zabka
authors.forename: Justyna Teresa
authors.initials: JT
authors.lastname: Polit
authors.forename: Janusz
authors.initials: J
authors.lastname: Maszewski
mesh_terms: D002453:Cell Cycle / Q000235:genetics*; D002467:Cell Nucleus / Q000235:genetics; D048429:Cell Size; D002843:Chromatin / Q000235:genetics; D019926:Cyclin B / Q000378:metabolism; D004249:DNA Damage; D004261:DNA Replication / Q000187:drug effects*; D018744:DNA, Plant / Q000235:genetics*; D003853:Deoxyribonucleosides / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006657:Histones / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D006918:Hydroxyurea / Q000494:pharmacology; D018519:Meristem / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D019697:Onions / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D010766:Phosphorylation; D010940:Plant Proteins / Q000378:metabolism*; D018517:Plant Roots / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D036226:Seedlings / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D002843:Chromatin; D019926:Cyclin B; D018744:DNA, Plant; D003853:Deoxyribonucleosides; D006657:Histones; D010940:Plant Proteins; D006861:Hydrogen Peroxide; D006918:Hydroxyurea