DNA Excision Repair and DNA Damage Checkpoints

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DESCRIPTION (provided by applicant): DNA damage is the predominant cause of cancer. The effect of DNA damage on humans may be prevented or ameliorated by cellular response mechanisms including DNA repair, DNA damage checkpoints, transcriptional reprogramming, and apoptosis. The goal of our research is to understand the mechanisms of DNA excision repair and DNA damage checkpoints in humans with the aim of providing mechanistic foundations for cancer prevention and treatment. To this end we will perform biochemical experiments to characterize these pathways. I. DNA EXCISION REPAIR. This repair system removes DNA damage caused by UV and by other chemical and physical agents that damage DNA. We have reconstituted this system in vitro. Using this defined system, we will investigate the damage recognition mechanism of human excision nuclease which has a wide range of substrates of dissimilar structures and uses ATP to achieve the requisite specificity. In addition, we will investigate the effect of histone modifications on excision repair in order to understand the roles of epigenetic effects on DNA repair. II. BIOCHEMICAL PROPERTIES OF HUMAN DNA DAMAGE CHECKPOINT PROTEINS. DNA damage checkpoints are signal transduction pathways that delay cell cycle progression to enable cells to escape the catastrophic consequences of replicating damaged DNA or segregating damaged chromosomes. In recent years, nearly two dozen proteins, including ATR-ATRIP, TopBP1, Rad17-RFC, the 9-1-1 complex, Timeless-Tipin, and Claspin have been identified by genetic analyses as essential for the checkpoint response to DNA damage by UV-mimetic agents. We will purify these human proteins and biochemically characterize them in terms of their structures, their interactions with one another, kinase activities, and DNA binding properties. These studies will provide the necessary information for the ultimate goal of reconstituting the ATR-mediated DNA damage checkpoint in vitro. III. ATR-DEPENDENT HUMAN DNA DAMAGE CHECKPOINT IN VITRO. Currently, there is no in vitro system that recapitulates the human DNA damage checkpoint response in its entirety. We will develop two in vitro systems for the ATR-mediated DNA damage checkpoint response. One system will be reconstituted from purified checkpoint proteins and damaged DNA. We will develop a second in vitro system in which the checkpoint response is activated by DNA gaps generated by nucleotide excision repair. The availability of such systems will provide biochemical tests of current checkpoint models that are based on genetic and cellular analyses and will establish well-defined systems for testing various bio- and chemotherapeutic strategies for cancer management.