DNA damage is either the causative or contributing factor in about 90 percent of human cancers. There are two cellular responses that counteract the untoward effects of DNA damage: removal of the damage by DNA repair pathways, including nucleotide excision repair, and activation of a DNA damage checkpoint response that blocks cell cycle progression so long as the DNA contains damage and hence prevents the harmful effects of replicating damaged DNA. Dr. Sancar's goal is to understand the molecular mechanism of excision repair and to investigate the biochemical basis of the DNA damage checkpoint response. He plans three general aims: I) Molecular Mechanism of Excision Repair. Excision repair encompasses dual incision/excision and repair synthesis. The excision step is carried out by 14 polypeptides in six repair factors and the reaction is stimulated by the XP-E gene product in vivo. With the exception of XPE, all genes known to participate in excision repair have been cloned and all repair factors except for TFIIH are available in recombinant form. It is proposed to clone the XPE cDNA and to define its function and to overproduce the six-subunit TFIIH in a baculovirus/insect cell vector/host system and to analyze its role in various steps of excision repair. The post incision step will be analyzed by identifying the proteins which remain associated with the excised and gapped DNA and by reconstitution of the repair synthesis step with purified DNA polymerases, RPA, RFC, and PCNA. Finally, the effect of chromatin structure on excision repair will be determined and the chromatin remodeling factors that modulate the accessibility of nucleosomal DNA to excision repair factors. II) Molecular Mechanism of Transcription-repair coupling. DNA lesions in the template strand of transcribed genes are repaired faster than lesions in the coding strand or in non-transcribed DNA. Using purified repair- and transcription proteins and naked DNA or DNA in minichromosomes, transcription-coupled repair will be reconstituted in vitro and the coupling mechanism will be elucidated. III) Biochemical analysis of DNA damage checkpoints. DNA damage causes transient arrest of cell cycle progression via biochemical pathways called DNA damage checkpoints. The biochemical properties of the proteins involved in this checkpoint response will be studied and an in vitro system for the reconstitution of DNA damage checkpoint pathways will be established.
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