This is the continuation of a project to study the operation of the nucleotidyl DNA excision repair pathway (NDERP) in vitro. In the first two years of the project we have characterized UV- dependent and non-specific endodeoxyribonuclease activities in permeable human fibroblasts using alkaline elution methodology to quantify DNA strand breaks. An ATP-dependent and UV- dependent endodeoxyribonuclease activity was observed in normal and xeroderma pigmentosum (XP) variant fibroblasts which represents the rate-limiting, dimer-directed incision step in the NDERP. This activity was undetectable in XP cells from complementation group A. A second UV-endodeoxyribonuclease activity which did not require ATP was identified in all human fibroblast strains which represents the redoxy endodeoxyribonuclease that cuts DNA at ring-saturated pyrimidines. Optimal conditions for assay of the UV- endodeoxyribonucleases were established. Various preparations of cellular extracts were added to permeable XPA to test for in vitro complementation of the defect in dimer-directed incision. UV-dependent incision could not be consistently demonstrated and non-specific endodeoxyribonuclease activities contaminated most cell extracts. Consequently, it appears that restoration of repair in permeable XPA will require fractionation of extracts to remove non-specific endodeoxyribonuclease. This fractionation will be facilitated by establishment of a plasmid-based endodeoxyribonuclease assay that can distinguish non-specific endodeoxyribonucleases from the two specific UV- endodeoxyribonucleases. A plasmid incision assay will be used to identify and isolate the dimer-directed UV-endodeoxyribonuclease from repair-proficient human cells. We will then determine whether incision-defective XPA also express this endodeoxyribonuclease. The isolated enzyme will be added to permeable XPA to test for restoration of dimer-directed incision of nuclear DNA. Peculiarities of reparative incision as demonstrated by other repair-deficient complementation groups of XP also will be examined. These studies will aid in identifying the bases of the various reparative defects seen in XP.