This revised version of the grant application is a proposal to continue studies of human Damage DNA Binding Protein (DDB). In previous studies, the applicant has shown DDB to be a heterodimer of p127 and p48 which has roughly the same damage preference as E. coli UvrA protein. In addition, missense mutations in p48 exist in - and only in - a subset of Xeroderma pigmentosum group E patients (XP-E Ddb-) whose protein lacks activity. Purified normal protein can correct the XP repair defect only when injected into Ddb- cells. In response to DNA damage, p48 but not p127 expression is increased. Finally, recent observations by Dr. Linn and others show that DDB interacts with several cellular and viral transcription transactivating proteins. Dr. Linn now proposes to explore the role(s) of the individual DDB subunits in DNA repair and/or transcription pattern changes following exposures to DNA damaging agents. In particular, he proposes to identify cellular components with which p127 and p48 interact. To accomplish this goal, he proposes to construct trans-hybrid mice lacking either p48 or p127 (or cell lines only if the mice are unviable). He will overexpress each subunit individually in insect cells to make antibodies and/or affinity columns to determine which protein(s) the subunits interact with. In addition to antibodies or DDB subunits as affinity column ligands, lectins specific for the DDB glycosylation may be utilized. Dr. Linn will also do yeast two and three hybrid screens and promoter sequencing to accomplish the same goal. He has made green fluorescent protein hybrid proteins with p48 and p127 and these, along with antibodies, will be used to look at cellular localization of these proteins and co-localization with any putative DDB-interacting proteins in both normal and XP-E cells. All interactions will be studied as a function of DNA damage exposure and cell cycle. He will continue to explore the significance of the interaction of DDB with the transcription factors EBNA2 and E2F1. Finally, he expects to obtain sequence data of the 5' un-translated region to look for motifs which could provide suggestions of functions for the peptides. The genome of S. pombe appears to code for a homologue of human p127 but not of p48. Moreover, while extracts of S. pombe do not contain DDB activity, such activity appears upon addition of human p48 to the extracts. Dr. Linn plans to use both genetic and biochemical analysis to find the molecular basis of this activity and the consequences of directed mutations in the putative p127 homologue.