The repair mode of DNA replication has been demonstrated in isolated nuclei from UV-irradiated human cells. Nuclei are incubated in a mixture containing [(3)H]
thymidine triphosphate and
bromodeoxyuridine triphosphate in a 1:5 ratio. The (3)H at the density of parental
DNA in alkaline
CsCl density gradients is then a measure of repair. In nuclei prepared from WI38 cells 30 min after irradiation, repair replication is UV dependent and proceeds at approximately the in vivo rate for 5 min. Repair replication is reduced in irradiated nuclei or in nuclei prepared immediately after irradiation. It is Mg(2+)-dependent and stimulated by added
ATP and deoxyribonucleoside triphosphates. No repair replication is observed in nuclei from
xeroderma pigmentosum (complementation group A) cells. However, upon addition of coliphage T4
endonuclease V, which specifically nicks DNA containing
pyrimidine dimers, repair replication is observed in nuclei from irradiated
xeroderma pigmentosum cells and is stimulated in WI38 nuclei. The reaction then persists for an hour and is dependent upon added
ATP and deoxyribonucleoside triphosphates. The repair label is in stretches of roughly 35
nucleotides, as it is in intact cells. Added
pancreatic DNase does not promote UV-dependent repair synthesis. Our results support the view that
xeroderma pigmentosum (group A) cells are defective in the incision step of the
DNA excision repair pathway, and demonstrate the utility of this system for probing DNA repair mechanisms.