Translesion replication in cisplatin-treated xeroderma pigmentosum variant cells is also caffeine-sensitive: features of the error-prone DNA polymerase(s) involved in UV-mutagenesis.
Patients with xeroderma pigmentosum variant (XP-V) have a higher risk to skin cancer and XP-V cells are extremely mutable by ultraviolet (UV). The defective gene encodes a DNA polymerase (Poleta) which catalyzed relatively accurate translesion synthesis past the cyclobutane dimer of UV-lesions instead of the replicative polymerase(s) that stalled just before the lesion. Pulse-chase studies have shown that translesion replication in XP-V cells is delayed, but does not completely cease. Taking these results together, error-prone polymerase(s) are plausively involved in the UV-mutagenesis in XP-V devoid of Poleta. However, less is known about the polymerase(s) in vivo. Using an alkaline sucrose density gradient centrifugation (ASDG) technique, translesion replication is detected in the two XP-V strains XP30RO and XP115LO. As reported by Lehmann et al. [Proc. Natl. Acad. Sci. U.S.A. 72 (1975): 219] in XP-V; (i) smaller replication products were accumulated after UV irradiation; (ii) the elongation of these products was delayed; (iii) the elongation was markedly inhibited by caffeine. XP-V cells UV-irradiated at mid-S phase were normally S-arrested, and no "override" by caffeine (i.e. abrogation of the S-checkpoint) was observed by flow cytometry, suggesting that caffeine does not act via cdc kinase here; (iv) butylphenyldeoxyguanosine (BuPGdR) inhibited elongation of replication products only in UV-irradiated XP-V cells; (v) dideoxycytidine or dideoxyinosine had no effect on this process in either normal or XP-V cells. Next, similar phenomena to UV (all of above i to v) were observed also in cisplatin-treated XP-V cells. Pol eta was indicated to participate in cisplatin-induced translesion replication in normal cells. Summing up the above results, the polymerase(s) which work in translesion replication in XP-V are probably BuPGdR-sensitive, insensitive to dideoxynucleotides and can bypass also cisplatin-lesions. To date, several polymerases capable of lesion-bypass synthesis have been isolated. The features presented here are quite useful for identifying the error-prone polymerase(s) involved in UV-mutagenesis.
Yamada
Kouichi
K
Division of Clinical Nutrition, The National Institute of Health and Nutrition, Shinjuku-ku, 162-8636 Tokyo, Japan. kouichi@nih.go.jp
Takezawa
Jun
J
Ezaki
Osamu
O
D002110:Caffeine / Q000494:pharmacology*; D002453:Cell Cycle; D002499:Centrifugation, Density Gradient; D002945:Cisplatin / Q000494:pharmacology; D004261:DNA Replication / Q000187:drug effects* / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D005347:Fibroblasts; D005434:Flow Cytometry; D006801:Humans; D016296:Mutagenesis / Q000187:drug effects; D013463:Sulfuric Acid Esters / Q000494:pharmacology; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics*; D000099270:Y-Family DNA Polymerases
D002110:Caffeine; D002945:Cisplatin; D004259:DNA-Directed DNA Polymerase; D013463:Sulfuric Acid Esters; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases; C007482:dimethyl sulfate