The objective of the proposed research is to identify and characterize the enzymes and reactions involved in excision-repair of DNA in human cells exposed to either radiation or alkylating agents. Efforts will continue to focus on the critical first step in excision, namely recognition of specific lesions by repair endonucleases or N-glycosylases. Previously, I have partially purified from cultured human lymphoblasts (CCRF-CEM line), an endonuclease acting specifically on DNA irradiated by UV or x-rays and an N-glycosylase acting specifically on 3-methyladenine in alkylated DNA. I now plan to purify both these enzymes from human placenta in quantities adequate for extensive characterization, and for use as reagents. Efforts will be made to identify the specific target-lesion recognized by the UVX-endonuclease. An immuno-specific assay will be developed for this enzyme, and cell extracts from individuals with "DNA-repair syndromes" will be screened for enzyme deficiency. Purified enzyme will be used as a specific probe for UVX-endonuclease sensitive sites in DNA from irradiated normal and mutant human fibroblasts to determine if the UVX-endonuclease or its target-lesion is implicated in radiation sensitivity. Similar studies already underway with lymphoblast enzyme will be carried out with purified placental 3-methyladenine-DNA glycosylase to determine if other target-lesions exist or if it is implicated in one of the human "DNA-repair syndromes." Both enzymes will be used to probe the kinetics, dose dependence and effects of inhibitors on repair of specific lesions in normal and mutant cells. Knowledge gained through this program will contribute to an understanding of the enzymatic mechanisms for excision repair, and also indicate their involvement in human genetic disorders associated with sensitivity to genotoxic agents and elevated incidences of cancer. Additionally, if resistance of tumors to radiation or radiomimetic drugs involves these excision pathways, this knowledge may well suggest modified approaches to cancer therapy.