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.