In order to preserve genomic stability, eukaryotic cells harbour highly flexible and integrated signalling systems that sense DNA damage and promote repair or cell death. Concomitantly, such signalling may modulate cell cycle progression to support the process of repair. The process of cell cycle arrest as a consequence of DNA damage signalling is known as the 'checkpoint response'. Activation of a DNA damage checkpoint can induce G1, S or G2 phase transient delay or trigger irreversible arrest such as senescence. Failure to monitor and to signal DNA damage is a hallmark of cancer cells.
Activating the DNA damage response
A regulatory network of proteins has been identified that participate in DNA damage checkpoint pathways. Central to this network are the ATM, ATR and the Mre11/Rad50/Nbs1 (MRN) complex. We are taking advantage of the vertebrate Xenopus laevis egg extract to study the biochemistry of the ATM, ATR and the Mre11 complex dependent DNA damage response. In the recent years we have established several assays based on cell free systems derived from Xenopus eggs to elucidate the biochemical bases of cell cycle checkpoints. We are using the Xenopus cell-free system in which we have already characterised ATM, ATR and the MRN complex to dissect the signal transduction pathway that senses DNA damage. Using this system we can characterise aspects of the checkpoint not easily accessible in other systems such as the study of the DNA lesions and the very early events responsible for the activation of the response. Among the cofactors required for ATM activation the MRN complex, which posses nuclease activity, plays a major role. Addition of DNA DSBs to Xenopus egg extract leads to MRN dependent ATM activation. Nuclease dependent resection of DSBs appears to be important for the activation of the DNA damage response We have obtained evidence suggesting that limited resection of DSBs is required to sustain ATM activity. In particular, we have shown that DSB processing depends on the endonuclease activity of the MRN complex, which resects ssDNA that progressively becomes available through the action of an associated helicase moving in the direction of the resection. As a result of this processing small single stranded DNA oligos (ssDNA oligos) are generated. These ssDNA oligos participate in the activation of ATM in response to DSBs (Figure 1). ssDNA oligos produced at DSBs probably interact with one or more subunits of the MRN complex that have DNA-binding domains and perhaps other ssDNA binding proteins such as SSB1 to promote continuous stimulation of ATM molecules.
Figure 1. In the presence of double strand breaks (DSBs) MRN promotes recruitment and activation of ATM molecules. Generation of ssDNA oligos by the MRN complexes enhances ATM activation.
Active MRN complexes can then facilitate the activation of inactive ATM molecules that have not yet engaged with DSBs amplifying the response. The creation of ssDNA oligos during the resection of DNA undergoing repair, either from 5' to 3' processing of DSBs or possibly from enlarging gaps in other forms of DNA repair is a unique signal of DNA damage. Whereas mononucleotides are produced by normal DNA metabolism, these ssDNA oligos form only during DNA damage processing and represent ideal alarm molecules that could work as second messenger signaling the presence of severe DNA damage. We are now testing the possibility to exploit this second messenger of DNA damage response to induce permanent cell cycle arrest such as senescence without damaging the genome. This strategy could be helpful in arresting the hyper-proliferation of cancer cells.
Identification of novel targets of the ATM/ATR dependent DNA damage response
The response to DNA damage is complex in vertebrates and some genes such as BRCA1 and p53 are present only in high eukaryotes. We are screening an expression library made of Xenopus laevis cDNAs to identify vertebrate targets of ATM and ATR. cDNAs can be in vitro transcribed and translated. Translated proteins are then mixed with extracts supplemented with damaged DNA, which is capable of activating ATM and/or ATR. Following incubation in extracts the protein mixture is separated on SDS-PAGE electrophoresis. Labeled proteins that undergo DNA damage dependent post-translational modifications are then isolated and characterised. This innovative strategy is allowing the rapid identification and cloning of proteins that are modified in the presence of active ATM and ATR. This screening has recently led to the isolation of the a centrosome protein that we named XCRA1 (Centrosome protein Regulated by ATM/ATR) as an ATM and ATR target involved in mitosis progression control following DNA damage. We have tested the effects of DNA damage occurring when cells are already in mitosis, a condition defined as mitotic catastrophe.
Xenopus egg extract is a powerful model system to study mitotic events such as spindle assembly. Addition of sperm nuclei to mitotic egg extract induces formation of half and full spindles. We found that ATM and ATR activation abolished spindle assembly inducing structures resembling aggregates formed by microtubules associated with chromosomal DNA. Inhibition of spindle assembly was dependent upon ATM and ATR. We then showed that XCRA1 is required for spindle assembly and that ATM/ATR dependent phosphorylation of XCRA1 inhibits spindle assembly. The checkpoint that we described operates in mitosis and might become important when other mechanisms preventing mitosis entry have failed. We now intend to characterise the molecular mechanism underlying the role of XCRA1 in spindle assembly. In particular, we would like to identify the proteins interacting with XCRA1 to see if the interaction is affected by the phosphorylation. Interestingly, human CRA1 is downregulated in several tumours suggesting that this protein is a novel tumor suppressor.
For a list of refereed research papers, see Publications (in navigation on left).
