DNA damage in somatic cells is one of the main causes of aging and many age-related diseases, including cancer and decreased tissue regeneration. Cells respond to DNA damage by triggering the checkpoint pathways that delay the cell cycle progression allowing time for DNA repair. The checkpoint pathway involves association of the PI3 kinases ATM/ATR with damaged chromatin and subsequent activation of the checkpoint kinases (Chk1 and Chk2) and p53, as well as other regulatory proteins that control the fate of a cell following DNA damage. Despite significant advances on the damage-response pathways, the mechanisms (sensors) that recruit ATM/ATR to damaged-DNA are not known. Our recent studies revealed that the damaged-DNA binding protein DDB functions as a damage-sensor that recruits ATR to the UV-damaged chromatin to activate the ATR-Chk1/p53 pathway. The objectives of this proposal are to further investigate the damage-sensor function of DDB and determine the spectrum of DNA damage for which DDB functions as a sensor leading to the activation of the ATR-checkpoint pathway. DDB is composed to two subunits: DDB1 and DDB2. The DDB2 subunit is transcriptionally induced by p53 upon DNA damage. We will test the hypothesis that the p53-induced expression of DDB2 is critical for a sustained activation of the checkpoints following DNA damage. Also, we plan to develop a knockout strain of mice lacking DDB1 to investigate the functions of DDB1 in a physiological context. Accumulating evidence indicates that the DDB1 subunit possesses functions that are independent of the DDB2 subunit. For example, DDB1 has been shown to be involved in the proteolysis of c-jun in conjunction with cullin 4A. Also, the fission yeast ortholgue of DDB1 has been implicated in the proteolysis through the ubiquitin-proteasome pathway. A conditional knockout strain of mice will be important in evaluating the various functions of DDB1 in a physiological context.
The specific aims are: 1. Does DDB function as a sensor for a wide range of DNA damages? Is the DDB-mediated activation of ATR-Chk1 linked to initiation of DNA replication? Does the DDB-mediated activation of ATR-Chk1 involve chromatin-remodeling by CBP/p300? 2.Does DDB play a role in the sustained activation of checkpoints following DNA damage? Does DNA damage-induced senescence depend upon DDB? 3. What are the in vivo functions of DDB1?
