Ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia Rad3-related (ATR) and the Mre11/Rad50/Nbs1 complex ensure genome stability in response to DNA damage. However, their essential role in DNA metabolism remains unknown. Here we show that ATM and ATR prevent accumulation of DNA double-strand breaks (DSBs) during chromosomal replication. Replicating chromosomes accumulate DSBs in Xenopus laevis egg extracts depleted of ATM and ATR. Addition of ATM and ATR proteins to depleted extracts prevents DSB accumulation by promoting restart of collapsed replication forks that arise during DNA replication. We show that collapsed forks maintain MCM complex but lose Pol e, and that Pol e reloading requires ATM and ATR. Replication fork restart is abolished in Mre11 depleted extracts and is restored by supplementation with recombinant human Mre11/Rad50/ Nbs1 complex. Using a novel fluorescence resonance energy transfer-based technique, we demonstrate that ATM and ATR induce Mre11/Rad50/Nbs1 complex redistribution to restarting forks. This study provides direct biochemical evidence that ATM and ATR prevent accumulation of chromosomal abnormalities by promoting Mre11/ Rad50/Nbs1 dependent recovery of collapsed replication forks.

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

DNA double strand breaks (DSBs) activate ATM and ATR dependent checkpoints that prevent the onset of mitosis. However, how cells react to DSBs occurring when they are already in mitosis is poorly understood. The Xenopus egg extract has been utilized to study cell cycle progression and DNA damage checkpoints. Recently this system has been successfully used to uncover an ATM and ATR dependent checkpoint affecting centrosome driven spindle assembly. These studies have led to the identification of XCEP63 as major target of this pathway. XCEP63 is a coiled-coil rich protein localized at centrosome essential for proper spindle assembly. ATM and ATR directly phosphorylate XCEP63 on serine 560 inducing its delocalization from centrosome, which in turn delays spindle assembly. This pathway might contribute to regulate DNA repair or mitotic cell survival in the presence of chromosome breakage.