Xeroderma pigmentosum cells with normal levels of excision repair have a defect in DNA synthesis after UV-irradiation.

Abstract

Cells cultured from most patients suffering from the sunlight-sensitive hereditary disorder xeroderma pigmentosum are defective in the ability to excise ultraviolet light (UV)-induced pyrimidine dimers from their DNA. There is, however,-one class of these patients whose cells are completely normal in this excision repair process. We have found that these cells have an abnormality in the manner in which DNA is synthesized after UVirradiation. The time taken to convert initially lowmolecular-weight DNA synthesized in UV-irradiated cells into high-molecular-weight DNA similar in size to that in untreated cells is much greater in these variants than in normal cells. Furthermore, this slow conversion of low to high-molecular-weight newly synthesized DNA is drastically inhibited by caffeine, which has no effect in normal cells. Recently, complementation studies have shown that there are several genetically different forms of XP, all deficient in excision-repair (3)(4)(5)(6)(7)(8). In addition there is a further class of XP patients (termed "XP variants" in this communication) which, while exhibiting the usual clinical symptoms, seem to be completely normal in excision-repair of pyrimidine dimers (9-14). All XP lines examined have normal rates of rejoining of DNA single-strand breaks induced by ionizing radiation (14).Despite very low levels of excision-repair in cells from most XP patients (and also in rodent cell lines), they can nevertheless tolerate the production in their DNA of over 105 pyrimi- In this communication we show that fibroblast cultures from three XP variants have normal levels of excision-repair, but are abnormal in postreplication repair. After UV-irradiation, the time taken for the newly synthesized DNA to attain a high molecular weight similar to that in unirradiated controls is much longer than in normal cells. Furthermore, this conversion of low-to high-molecular weight DNA is drastically inhibited by caffeine, which has very little effect in normal human cells. MATERIALS AND METHODSCell Lines used in these experiments were primary fibroblasts from healthy donors and XP patients listed in Table 1.Excision of Pyrimidine Dimers Measured by Loss of UVEndonuclease-Susceptible Sites. This procedure has been described in detail (24). Briefly; fibroblast cells cultured as described in ref. 24 were labeled with [3H]thymidine, UVirradiated, and incubated in the absence of radioactive label for various times. They were then mixed with an equal volume of unirradiated cells whose DNA had been labeled with [14C]-dT. DNA was extracted from the mixed cell population and incubated with or without the UV-specific endonuclease from Micrococcus luteus, which'specifically nicks DNA near pyrimidine dimers (24,25). The DNA products resulting from enzymic attack were centrifuged through 5-20% alkaline sucrose gradients at 40,000 rpm for 135 min at 210 in an SW56 rotor. After centrifugation, fractions were collected, radioactivity was determined, and the weight-average molecular weight of the DNA distributions ...

DNA repair, DNA replication and human disorders: A personal journey

“…Additionally I and others had shown that caffeine inhibited PRR in rodent cells [21]. Remarkably, we found that it had no effect on PRR in normal human fibroblasts, but completely inhibited the already deficient PRR in XPV fibroblasts, thus generating a huge difference between the normal and XPV cells [22]. In 1970s, effects of caffeine were interpreted in terms of its inhibitory effects on cyclic AMP phosphodiesterase, whereas these days everybody thinks of it as an inhibitor of ATR and ATM.…”

“…The data from that blind experiment are shown in Fig. 4 of the paper that was published in PNAS in 1975 (see Fig 2B) [22] and is my most cited paper. We then built on this work and spent a lot of time in the next few years debating in the literature and at meetings whether the replication fork was blocked at lesions, whether it restarted beyond the lesion after a delay, or whether synthesis bypassed the lesion directly without discontinuities (e.g.…”

p53 suppression overwhelms DNA polymerase η deficiency in determining the cellular UV DNA damage response

“…In XP-V cells that lack the translesional DNA Pol η, UV damage to DNA causes an asymmetric replication fork arrest due to a replication block on the leading strand of DNA [41][42][43][44][45][46]. These structures trigger the activation of intra-S-phase checkpoints [44].…”

Translesion synthesis in mammalian cells

“…Most XP patients are deficient in the ability to remove UV photoproducts from their DNA by nucleotide excision repair (NER), but about 20 % are normal in this respect and have problems in replicating their DNA after UV-irradiation [5]. The gene defective in these XP variants encodes polη.…”