An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63

Abstract

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. 3

Interplay between the DNA Damage Proteins MDC1 and ATM in the Regulation of the Spindle Assembly Checkpoint

“…Several studies indicate that the SAC can be activated by exogenous DNA damage (56 -58) and that ATM can activate the SAC due to excessive DNA damage in mitosis (21,59). We cannot distinguish whether the role of ATM and MDC1 in SAC activation is due to a DNA damage-independent function of these DDR proteins or that the SAC is activated by the DDR.…”

“…The active form of ATM localizes at centrosomes during mitosis (19,20). When excessive DNA damage is introduced to mitotic cells, ATM inhibits centrosome-dependent spindle formation, leading to a transient mitotic arrest, probably through SAC activation (21). Additionally, following spindle disruption, ATM relocalizes from centrosomes to distinct cellular foci, which also contain activated p53 (22).…”

New insights into the regulation of anaphase by mitotic cyclins in budding yeast

“…Moreover, the stronger requirement of Clb2 in a rad51Δ context is reasonable, since it has been suggested that SPB regulation is altered by the DNA damage response. [60][61][62][63] Mitotic cyclins could phosphorylate substrates upon end-on attachment that localize at SPB, switching the balance of MTs dynamic toward growth suppression. A recent study in Hela cells has shown that mitotic cyclinB1 localizes to kinetochores and plays an important role in efficient attachment between kinetochore and microtubule during prometaphase.…”

Induction of ASAP (MAP9) contributes to p53 stabilization in response to DNA damage

“…21,22 Similarly, ATM/ATR and Chk1/ Chk2 kinases are localized at centrosomes 23 and have recently been shown to control mitotic events and centrosome-dependent spindle assembly. 24 Here we propose a novel mechanistic pathway by which ASAP modulates p53 functions. Following DNA damage, ASAP is transiently stabilized.…”