Sunlight-induced skin cancer is a major public health concern. Since the discovery of structural damage in UV-irradiated DNA 25 years ago, the molecular mechanisms underlying photocarcinogenesis have been a major focus of investigation. And yet, the molecular events that mitigate the effects of UV damage in DNA, and determine their pathogenic consequences in higher eukaryotes, are not well understood. Immunological and biochemical techniques have revealed a diverse spectrum of photodamage in DNA; each lesion may have its own cytotoxic and mutagenic potential and may be modulated by distinct DNA repair mechanisms. Recently, new approaches have been developed to measure DNA repair in specific sequences and have been used to describe the preferential excision of cyclobutane dimers in transcribing genes. As data accumulate, it is evident that many factors affect DNA repair in specific genes and that other photoproducts, in addition to the cyclobutane dimer, may be biologically important. In this regard, the (6-4) photoproduct has gained much attention over the past few years as a significant lethal and mutagenic determinant in UV-irradiated DNA. However, little is known regarding the induction and repair of this lesion in specific regions of mammalian chromatin. It is our goal to develop new approaches and exploit existing techniques to quantify (6-4) photoproducts in human and rodent cells. We will adapt the technique of Bohr and Hanawalt to the analysis of (6-4) photoproducts in DNA as """"""""photoinduced alkali-labile sites"""""""" (PALS) using Southern transfer and hybridization to specific probes. In addition, we will modify this procedure to quantify (6-4) photoproducts in specific sequences as antibody-binding sites using a mobility shift immunoassay. We will use these techniques to evaluate preferential (6-4) photoproduct repair in mammalian genes and investigate how DNA structure and function affect its formation and excision. In addition, we will use radioimmunoassays to compare the kinetics of (6-4) photoproduct repair in functionally-defined regions of DNA (i.e., nuclear matrix and DNase I-sensitive domains of chromatin). Along with established techniques for analyzing cyclobutane dimers in specific DNA sequences, these novel procedures will be used to investigate the function of the human ERCC2 gene and determine its role in mammalian DNA excision repair. By characterizing the fine structure of (6-4) photoproduct induction and repair in mammalian cells, we hope to elucidate the lethal and mutagenic mechanisms of UV light and further our understanding of the molecular basis of carcinogenesis.
