Smith, E.A. (2010) An ATM and ATR dependent checkpoint inactivates spindle assembly by targeting CEP63. Doctoral thesis, UCL (University College London).
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The effects of ATM and ATR signalling induced by chromosomal breakage have been described extensively in modulating cell cycle progression up to the onset of mitosis. However, DNA damage checkpoint responses in mitotic cells are not well understood. This thesis reports on the effects of double strand breaks on the progression of mitosis. We found ATM and ATR activation can occur in mitotic Xenopus laevis egg extract and the induction of ATM and ATR by chromosomal breakages inhibits spindle assembly in both Xenopus egg extract and somatic cells. The delay in mitotic progression induced by ATM and ATR was found not to involve major spindle assembly factors activities such as, Cdk1, Plx1 and RCC1/Ran-GTP. However, normal anastral spindles formation around linear DNA coated beads, which can activate ATM and ATR, linked centrosome-driven spindle assembly to ATM and ATR dependent spindle defects. cDNA expression library screening was undertaken to identify novel ATM and ATR targets in this mitotic checkpoint pathway, through which the novel centrosomal protein XCEP63 was identified as a likely candidate. Data obtained from depletion and reconstitution of XCEP63 in Xenopus egg extract established that normal centrosome-driven spindle assembly requires XCEP63. Moreover, ATM and ATR phosphorylates XCEP63 on serine 560 and promotes delocalisation from the centrosome. ATM and ATR inhibition or addition of non-phosphorylable XCEP63 recombinant protein mutated at serine 560 prevents spindle assembly abnormalities. These findings suggest that ATM and ATR regulate mitotic events by targeting XCEP63 and centrosome-dependent spindle assembly. This pathway may provide support for DNA repair processes or regulate cell survival in the presence of mitotic DNA damage.
|Title:||An ATM and ATR dependent checkpoint inactivates spindle assembly by targeting CEP63|
|Open access status:||An open access version is available from UCL Discovery|
|Additional information:||Section entitled: Supplemental Figure Legend and Supplementary Materials and Methods (17 pages) not included in pdf copy|
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