Cell-free repair of UV-damaged simian virus 40 chromosomes in human cell extracts. II. Defective DNA repair synthesis by xeroderma pigmentosum cell extracts.
We have constructed a cell-free DNA repair system with UV-irradiated SV40 minichromosomes, as described in the accompanying paper (Sugasawa, K, Masutani, C., and Hanaoka, F. (1993) J. Biol. Chem 268, 9098-9104). In this study, we examined DNA repair synthesis by cell extracts from seven xeroderma pigmentosum (XP) complementation groups, A through G. DNA repair synthesis by XP cell extracts was lower than that with repair-proficient human 293 cell extract and did not increase to the level with the latter on increase in the amount of cell extract or the incubation time. The defects of XP cell extracts were complemented by addition of extracts from cells of different complementation groups, indicating that defective proteins in XP-A through G cells are directly involved in DNA repair. Addition of T4 endonuclease V, which is reported to complement defects of XP cells, stimulated DNA repair synthesis by the 293 cell extract, and also complemented the defects of all XP cell extracts. The XPAC gene product was shown to be involved in DNA repair synthesis using anti-xpac serum and xpac protein produced in Escherichia coli. Anti-xpac serum inhibited DNA repair synthesis by the 293 cell extract and xpac protein reversed the inhibition. Furthermore, xpac protein complemented the defects of extracts of two lines of XP-A cells (XP2OSSV and XP12ROSV) but had no effect on the reactions of extracts from cells of other complementation groups. These findings are consistent with previous results obtained in experiments with cells, indicating that our system is useful for analyzing the mechanisms of DNA excision repair in mammalian cells.
Masutani
C
C
Cellular Physiology Laboratory, Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
Sugasawa
K
K
Asahina
H
H
Tanaka
K
K
Hanaoka
F
F
D002460:Cell Line; D002461:Cell Line, Transformed; D002474:Cell-Free System; D002875:Chromosomes / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics* / Q000528:radiation effects; D005816:Genetic Complementation Test; D006801:Humans; D013539:Simian virus 40 / Q000235:genetics*; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics*
D004279:DNA, Viral