Nucleotide excision repair (excision repair) is the most general DNA repair system which protects humans from DNA damage-induced cancers. It removes all types of lesions from DNA. Defective excision repair causes xeroderma pigmentosum (XP), a hereditary disease characterized by very high incidence of actinic cancers and frequently with neurological abnormalities. More importantly, excision repair is the moor repair mechanism for lesions caused by the two most common carcinogens: sunlight and cigarette smoke. Genetic and biochemical data indicate that 16 polypeptides are directly involved in the first step of excision repair which Is the removal of the lesion in a 27-29 nt-long oligomer. Following excision the repair gap is filled in by replication proteins and ligated. Our goal is to understand the molecular mechanisms of these phenomena and their relationships to other cellular reactions. We will conduct the following experiments. I. Purification of Excision Repair Proteins. The proteins will be purified from their natural source for analytical studies. For detailed mechanistic and physical chemical investigations we will express the repair proteins in heterologous overproducing systems. II. Mechanism of Excision. By using both natural and recombinant proteins, the order of assembly of the 16 polypeptides (in 6 protein complexes) at the damage site, the structure of the DNA in the pre- and postincision complexes and the protein compositions of these complexes will be determined. Using protein engineering techniques the domains and amino acid residues involved in intersubunit and DNA interactions, DNA unwinding, and dual incisions will be identified. III. Repair Synthesis. Following the dual incisions, replication proteins displace the repair synthesis proteins fill In the gap and ligate the patch. We will identify the proteins involved in this reaction and determine the protein-protein interactions involved in this step. IV. Transcription-Repair Coupling. Employing both cell-free extracts and defined transcription and repair systems we will determine how a stalled RNA Pol Il specifically targets the excision nuclease to the lesion in the template strand. V. Excision Repair and Cellular Response to DNA Damage. Genotoxic stress induces cell cycle arrest, transcriptional Induction and posttranscriptional modification of several proteins. The interrelationships of these phenomena with excision repair will be studied. In particular, the effects of damage binding HMG-domain proteins, and the p53 tumor suppressor on excision repair and the effect of repair protein phosphorylation on cellular response to DNA damage will be investigated with the ultimate goal of devising mechanism-based chemotherapeutic strategies.
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