Vincenzo Costanzo : DNA Damage and Genomic Stability

Goals

Previous and current research

Cells respond to DNA damage by activating a multi-faceted response called DNA damage response. Defects in the DNA damage responses can result in genomic instability. Several cancer-prone syndromes reflect defects in the specific genes of the DNA damage response. They include Ataxia-Telangiectasia or A-T, Nijmegen Breakage Syndrome and A-T Like Disorder or A-TLD. We have established a cell-free system based on Xenopus laevis eggs extracts that recapitulates several aspects of the DNA damage response, including the DNA damage checkpoint and the activation of DNA repair. This is the first cell-free system that recapitulates the DNA damage response in vitro and allows biochemical studies of its different steps. Using this system we have identified novel DNA damage checkpoints that prevent initiation of DNA replication following DNA damage (Costanzo et al., 2000, 2003). This checkpoint signals entirely through Xenopus ATM (X-ATM) and Xenopus ATR (X-ATR). Initiation of DNA replication requires the assembly of a pre-replicative complex (pre-RC) on chromatin, which subsequently allows the binding of Cdc45 protein that is critical for polymerase loading. Initiation also requires the concerted activity of the cell cycle regulated protein kinases such as Cdk2 and Cdc7. We have shown that ATM and ATR dependent signalling leads to the rapid p53-independent inhibition of Cdk2 and Cdc7/Dbf4. (Costanzo et al., 2000, 2003).

In addition, we have used this cell-free system to study DNA repair pathways in vitro. We have identified a novel function for Mre11 that accounts for its essential role in vertebrates (Costanzo et al., 2001, 2004). In the absence of Mre11, DNA double strand breaks (DSBs) accumulate during normal DNA replication, rapidly leading to multiple chromosomal abnormalities. These findings demonstrated that DSBs normally occur during unchallenged chromosomal DNA replication and that the function of Mre11 complex is to prevent their accumulation. More recently we have established that Mre11 complex is required for ATM activation in presence of DSBs (Costanzo et al., 2004).

Future projects

Using this system we intend to study the biochemistry of DNA damage response and the coordination of the DNA damage response with the repair of defined DNA lesions during the cell cycle. In particular, we intend to:

1.Dissect the signal transduction pathway that senses DNA damage and promotes cell cycle arrest and DNA damage repair;

2.Analyse the coordination of DNA replication, DNA damage repair and DNA damage checkpoint at molecular level;

3.Identify novel substrates of the ATM/ATR dependent DNA damage response;

4.Identify small molecules that modulate the DNA damage response