Research in this laboratory is concerned with understanding the biology of radiation-induced DNA damage and its repair, and the mechanisms by which unrepaired damage is translated into biologic effects such as cell lethality, mutation, and carcinogenesis. Particular attention is given to the relationship of DNA damage and repair to the clinical manifestations of human genetic disorders [i.e. xeroderma pigmentosum (XP), Cockayne's syndrome (CS)] that exhibit cellular hypersensitivity to ionizing and nonionizing radiations, mental and growth retardation, and a predisposition to cancer. The broad and long term objective of this proposal is to gain insight as to the biologic significance of the cell's nuclear architecture on the regulation of DNA repair in vivo. In contrast to the significant progress in understanding the biochemical components and their interactions of DNA repair in vitro, very little is known about the nature of these interactions in vivo. Furthermore, there is very limited information about the cellular regulation of DNA repair and how interactions of DNA repair proteins with components of the nuclear architecture influences DNA repair in vivo.
The specific aims of this study are: 1) to confirm the role of intracellular trafficking of XPG proteins in regulation of DNA repair, 2) to understand interactions between DNA repair proteins and intranuclear architectural components, 3) to identify intranuclear components that regulate XPG activity at the cellular level, 4) to explore the molecular mechanism that controls the UV-induced movement of XPG and transcription-coupled repair. The experimental design to be used in the pursuit of these aims will employ: 1) deletion-mutagenesis to confirm the nuclear localization signals in XPG and to evaluate the effect of structural context for efficient nuclear targeting, 2) point-mutagenesis to determine the role of phosphorylation on nuclear localization, 3) biochemical fractionation and purification of intranuclear structures that control DNA repair at the cellular level, 4) in vitro phosphorylation assay to evaluate the role of phosphorylation in the regulation of UV-induced movement of XPG in the nucleus, 5) molecular cloning to identify intranuclear structural entities that control DNA repair activity in vivo, and the kinases that are postulated to control the cellular activity of XPG protein. Results of this research will help the researchers to understand the link between DNA repair and transcription at the cellular level, which appears to be the basis for the complexity of the XPG/CS syndrome.
