Microinjection of T4 endonuclease V produced by a synthetic denV gene stimulates unscheduled DNA synthesis in both xeroderma pigmentosum and normal cells.
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.
Yamaizumi
M
M
Institute for Molecular and Cellular Biology, Osaka University, Japan.
Inaoka
T
T
Uchida
T
T
Ohtsuka
E
E
D000818:Animals; D002478:Cells, Cultured; D004260:DNA Repair; D004274:DNA, Recombinant; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000008:administration & dosage*; D005814:Genes, Viral; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008845:Microinjections; D013604:T-Phages / Q000235:genetics; D014466:Ultraviolet Rays; D014764:Viral Proteins; D014983:Xeroderma Pigmentosum / Q000235:genetics*
D004274:DNA, Recombinant; D014764:Viral Proteins; D004706:Endodeoxyribonucleases; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer)