Drosophila replication and repair proteins: Proliferating cell nuclear antigen (PCNA)

Proliferating cell nuclear antigen (PCNA), a protein intimately involved in both replication and repair, has been identified in eukaryotes at all leve ls of evolution. Is primary sequence, Drosophila melanogaster PCNA is 73% i dentical to mammalian PCNA. Moreover, it is able to substitute for mammalia n PCNA in at least one intricate cell-free replication assay. Mutations in the gene for Drosophila PCNA, including some that are temperature sensitive , have been reported. Procedures are described for the biochemical purifica tion of wild-type PCNA from a population of 6- to 18-h-old Drosophila embry os. Procedures were also developed for purification of unmodified wild-type Drosophila PCNA after induction of expression in Escherichia coil. An NH2- terminally His-tagged but otherwise wild-type Drosophila PCNA, as well as m utant His-tagged PCNA, were also engineered and purified to apparent homoge neity. Finally, an in situ polyacrylamide gel technique allows DNA polymera se assays to be performed on portions of single adults as well as single Dr osophila embryos. This assay should tremendously facilitate systematic gene tic studies of metazoan replication and repair. (C) 1999 Academic Press.