Xeroderma pigmentosum variant cells are less likely than normal cells to incorporate dAMP opposite photoproducts during replication of UV-irradiated plasmids.

Abstract

Xeroderma pigmentosum (XP) variant patients show the clinical characteristics of the disease, with increased frequencies of skin cancer, but their cells have a normal, or nearly normal, rate of nucleotide excision repair of UV-induced DNA damage and are only slightly more sensitive than normal cells to the cytotoxic effect of UV radiation. However, they are significantly more sensitive to its mutagenic effect. To examine the mechanisms responsible for this hypermutability, we transfected an XP variant cell line with a UV-irradiated (at 254 nm) shuttle vector carrying the supF gene as a target for mutations, allowed replication of the plasmid, determined the frequency and spectrum of mutations induced, and compared the results with those obtained previously when irradiated plasmids carrying the same target gene It is now widely recognized that the transformation of normal cells into tumorigenic cells is a multistep process, and substantial evidence indicates that mutations play a fundamental role in cellular transformation and carcinogenesis, as well as in many inheritable diseases and developmental anomalies (1, 2). However, our understanding of the factors and influences governing the formation of these changes in gene structure is considerably less advanced. Cells isolated from patients with the rare autosomal recessive disorder xeroderma pigmentosum (XP) present a unique model system for investigating DNA repair and mutagenesis in human cells. In the present study, we made use ofa shuttle vector assay to investigate the kinds of mutations induced when a UV-irradiated plasmid replicated in cells derived from the class of XP patients called XP variants in order to provide clues to the mechanisms responsible for the hypermutagenic effect of UV radiation on these cells. XP variants inherit the characteristic predisposition to sunlight-induced skin cancer, but unlike the majority of XP patients, their cells do not exhibit a significant deficiency in the rate of nucleotide excision repair of endogenous UVinduced DNA damage, including both cyclobutane pyrimidine dimers (3-8) and pyrimidine-pyrimidone(6-4) photoproducts (7, 8). Cells from XP variant patients have an abnormality in the manner in which DNA replicates on a template containing UV lesions (9-11) and an inability to convert a very minor UV photoproduct to an excisable lesion (12). They are only slightly more sensitive than cells from normal donors to the cytotoxic effect of UV but are significantly more sensitive to its mutagenic action (13-15). However, the molecular mechanism(s) responsible for the abnormal sensitivity of XP variant cells to UV-induced mutations has not been explained.To examine this question, we UV-irradiated a shuttle vector, pS189 (16), carrying the supF gene as the target for mutations and transfected the plasmids into a simian virus 40-transformed XP variant cell line (XP-V) where they could be replicated by the human cell polymerase(s). The progeny plasmids were analyzed for the frequency of supF mutants and the kinds of mu...

Evidence that in Xeroderma Pigmentosum Variant Cells, which Lack DNA Polymerase η, DNA Polymerase ι Causes the Very High Frequency and Unique Spectrum of UV-Induced Mutations

“…C transitions (10). This higher frequency and altered spectrum of UV-induced mutations was also observed in a target gene of UV-irradiated plasmids replicated in cells from XPV patients, compared with what was found when the plasmid replicated in normal human cells (11). A similar high frequency and unusual spectrum of UV-induced mutations was found when replication-competent cell-free extracts from XPV cells were used to replicate a target gene, compared with such extracts from normal cells (12).…”

DNA Replication in the Face of (In)surmountable Odds

“…[24][25][26][27] Mutations in Pol H are found in xeroderma pigmentosum (XP-V) cells 28,29 and cause enhanced arrest of DNA replication at pyrimidine dimers and increased mutagenesis. [33][34][35][36] These mutations cause severe truncations of the protein, or prevent the enzyme from relocating to replication foci. [30][31][32] Pol H can replicate many kinds of DNA lesions, and preferentially inserts As opposite thymine-containing lesions contributing to accurate replication of UV-induced pyrimidine dimers.…”

Deficiency of the Caenorhabditis elegans DNA Polymerase .ETA. Homologue Increases Sensitivity to UV Radiation during Germ-line Development

“…Most TLS polymerases belong to the recently discovered Y-family (Ohmori et al, 2001). After UV irradiation, cultured cells from XP-V patients are defective in DNA replication and exhibit an increased mutation frequency with an altered spectrum (Lehmann et al, 1975;Maher et al, 1976;Wang et al, 1991Wang et al, , 1993Waters et al, 1993;Stary et al, 2003). The XPV gene encodes human polh (Masutani et al, 1999a;Johnson et al,1999) which bypasses CPDs by incorporating correct nucleotides (Masutani et al, 1999b(Masutani et al, , 2000Johnson et al, 2000;Washington et al, 2001;Kusumoto et al, 2004;McCulloch et al, 2004), indicating that Polh is involved in the relatively accurate TLS pathway for bypass of UV-induced lesions in vivo and in vitro.…”