Vincenzo Costanzo

DNA damage and genome stability: studying the role of ATM, ATR and the Mre11 complex in vertebrate organisms.

DNA is continuously subjected to exogenous and endogenous damaging insults. DNA damage must be detected in order to prevent loss of vital genetic information.

Cells respond to DNA damage by activating checkpoint pathways that delay progression through the cell cycle. These signalling pathways operate throughout the cell cycle. Activation of a DNA damage checkpoint can induce G1, S or G2 phase transient delay, trigger irreversible arrest such as senescence, promote DNA repair or induce cell death. Failure to monitor and to signal DNA damage is a hallmark of cancer cells.

A regulatory network of proteins has been identified that participate in DNA damage checkpoint pathways. Central to this network are ATM, ATR and the Mre11/Rad50/Nbs1 complex. When mutated, ATM is responsible for Ataxia Telangiectasia (A-T), an autosomal recessive disease that displays a complex phenotype. Patients exhibit increased risk for cancer and radiation sensitivity. ATM is a serine/threonine protein kinase that senses the damage of DNA and activates a pathway that leads to the phosphorylation of proteins such as Brca1, Nbs, Chk1, Chk2 p53 and many others. All these genes have been shown to be involved in human cancer when mutated.

ATM is prevalently activated by Double-Strand Breaks (DSBs). DSBs are among the most harmful form of DNA damage. They can indeed lead to chromosome breaks and rearrangements with consequent loss of genetic information. ATR is a kinase similar to ATM. The difference with ATR is that it responds to single strand DNA gaps and is essential for life. Decreased levels of ATR protein lead to Seckel disease.

Cells derived from Seckel syndrome patients have spontaneous chromosomal aberrations and DNA repair defects. Crucial for ATM activation and for DNA repair is the Mre11/Rad50/Nbs1 complex. Although the players of DNA damage response are well known, little is known about the biochemical mechanisms underlying their function. The detailed biochemical analysis of the DNA damage response has been impeded by the lack of cell-free systems recapitulating this process in vitro.

We have set up several assays to study the biochemistry and the biology of DNA damage response using cell free extracts derived from the eggs of the vertebrae organisms Xenopus laevis. This model system is a unique tool to investigate the molecular aspects underlying cell cycle events such as DNA replication and mitosis in the test tube. Major discoveries in the cell cycle field have been made using this model system. We are now taking advantage of this system to study the biochemistry of the ATM, ATR and the Mre11 complex dependent DNA damage response. Discoveries made with this model organism will then be translated into human cells.

Using the cell free system we are interested in the following aspects of the DNA damage response:

1. Studying the role of ATM, ATR and the Mre11 complex in preventing genome instability during chromosomal DNA replication.
2. Identifying novel ATM and ATR targets and pathways that contribute to maintain genome stability during cell cycle.
3. Studying the very early events leading to ATM activation.