A structural gene for
T4 endonuclease V was constructed by ligating synthetic
oligonucleotides. The
endonuclease V was overproduced in E. coli under control of the E. coli
tryptophan promoter and purified to apparent homogeneity. The product had comparable
DNA glycosylase and apurinic/apyrimidinic (
AP) endonuclease activities to the natural
enzyme in vitro. When this
endonuclease V was microinjected into the cytoplasm of
xeroderma pigmentosum (XP) cells of complementation group A, B, C, D, F, G or H, unscheduled
DNA synthesis (UDS) above the residual level was detected in all the cells at a dose of about 10(3) molecules following UV irradiation. The gain numbers of UDS in these XP cells increased with increase in the dose of
enzyme and reached a plateau at the normal cell level on introduction of about 10(4) molecules. Introduction of more
enzyme into either XP cells or normal human cells did not increase the grain number under regular labelling conditions (2.5 h, 37 degrees C). In normal mouse cells, introduction of the
enzyme increased the grain number more than 4-fold under the same conditions during at least 8.5 h following UV irradiation. Furthermore, with a labelling time of 30 min, the
enzyme more than doubled the grain number even in normal human cells.