Vincenzo Costanzo

DNA damage and genomic stability: the role of ATM, ATR and Mre11

Cells respond to DNA damage by activating a multi-faceted process called DNA damage response. Failure to monitor and to signal DNA damage leads to genomic instability, the hallmark of cancer cells.

Several cancer-prone syndromes reflect defects in the specific genes of the DNA damage response. They include Ataxia-Telangiectasia or A-T, in which the ATM gene is mutated, Nijmegen Breakage Syndrome and A-T Like Disorder.

ATM deficient cells exposed to ionizing radiation cannot prevent DNA replication and undergo Radio-resistant DNA Synthesis (RDS) leading to genomic instability. By blocking ATM function we have demonstrated the existence of an ATM-dependent and p53 independent DNA replication block in Xenopus egg extracts exposed to DNA molecules containing double strand breaks (DSBs) (Costanzo et al. 2000). In addition, we have shown that several DNA damage responses and DNA damage repair pathways can be easily activated in Xenopus eggs extracts (Costanzo et al. 2001, Costanzo et al. 2003).

A multi-faceted approach will be used which includes the use of the powerful cell free systems based on Xenopus laevis egg extracts, proteomics, mammalian cell cultures, somatic gene knock out/ knock down, yeast two-hybrid, molecular biology and classical biochemistry.


Activation of DNA Damage Response
We will characterize aspects of the checkpoint not easily accessible in other systems such as the analysis of the type of DNA lesions and the very early events in the activation of the DNA damage response. The Xenopus cell-free system will allow the study of the biochemistry of the major players of the DNA damage response such as ATM, ATR and Chk1/Chk2 that lead to DNA replication arrest through down-regulation of Cdc25-Cdks and DDks activities.


Coordinating the DNA damage response
We will study the coordination at molecular level of the DNA damage checkpoint initiated by ATM/ATR and the DNA damage repair operated by Mre11 complex and other repair genes. We will test the hypothesis that some of the proteins important for DNA damage checkpoint and DNA repair are also essential to prevent the accumulation of DNA damage during unchallenged chromosomal replication and will give rise when depleted or inactivated to damaged DNA. We will investigate the role of proteins implicated in maintaining genomic stability such as ATR, BRCA1, BRCA2 and Rad51 in chromosomal DNA replication. In particular, we will study their role in replication initiation, replication elongation and replication fork stability, using the assays that we developed such as in vitro DNA replication, TUNEL analysis and chromatin binding of initiation and replication factors.


Identification of novel targets of ATM/ATR
Finally, we will search for novel proteins that are involved in the DNA damage response. We will screen expression 'small pools' libraries for translated proteins that undergo post-translational modification, following exposure to extracts containing DSBs. We have already shown that DSBs induce phosphorylation and mobility changes in H2ax and Mre11 (Costanzo et al. 2001). In addition, we have shown that introduction of DSBs in Xenopus laevis egg extracts induces the phosphorylation of several proteins detected by the anti SQ/TQ ATM/ATR substrate.

Using these combined strategies we will rapidly identify and clone proteins that are modified in the presence of DSB. Some of the gene products identified will be further studied and used as probes to isolate the human homologue. In addition, bioinformatics tools will be used to evaluate the potential biological role of identified ATM/ATR substrates. The most interesting genes with a clear role in the DNA damage response will be evaluated for mammalian knock-out studies.


The successful candidates will be involved in an exciting area of research focused on the physiological processes responsible for the maintenance of genomic stability that is lost in cancer cells.

References

1. Costanzo et al. Mol Cell 2000, Sep; 6(3): 649-59
2. Costanzo et al. Mol Cell 2001, Jul; 8(1):137-47
3. Costanzo et al. Mol Cell 2003, Jan; 11(1): 203-13