Restoration of ultraviolet-induced unscheduled DNA synthesis of xeroderma pigmentosum cells by the concomitant treatment with bacteriophage T4 endonuclease V and HVJ (Sendai virus).

Abstract

Ultraviolet (UV)induced unscheduled DNA synthesis of xeroderma pigmentosum cells, belonging to complementation groups A, B, C, D, and E, was restored to the normal level by concomitant treatment of the cells with T4 endonuclease V and UV-inactivated HVJ (Sendai virus). The present results suggest that (1) T4 endonuclease molecules were inserted effectively into the cells by the interaction of HVJ with the cell membranes, (I) the enzyme was functional on human chromosomal DNA which had been damaged by UV irradiation in the viable cells, (3) all the studied groups of xeroderma pigmentosum ("variant" was not tested) were defective in the first step (incision) of excision repair. (5) suggested that XP cells fail to start the excision process because they lack the required function of an UV-specific endonuclease. Paterson et al. (6) and Buhl and Regan (7) reported that an UV-specific endonuclease from Micrococcus luteus was effective for introducing single-strand breaks in UV-damaged DNA of XP cells (both classical and deSanctis-Cacchione). On the contrary, Cleaver (8) observed that after UV-irradiation strand breaks accumulated in DNA of XP cells (deSanctis-Cacchione). All these experiments (5-8) were carried out using an alkaline-sucrose gradient centrifugation technique in vitro to estimate the degree of strand breaks of UV-irradiated DNA.We planned to identify autoradiographically what kind of cells in the five complementation groups was defective in the enzyme for strand breaks of UV-damaged DNA, by insertion of an UV-specific endonuclease into viable XP cells. HVJ is well known to have cell fusion activity (9). At an early stage of the cell fusion reaction, the structure of the Abbreviations: HVJ, hemagglutirating virus of Japan, synonym: Sendai virus; XP, xeroderma pigmentosum; UV, ultraviolet; PEME, phosphate-ethylene glycol-mercaptoethanol-EDTA buffer; ENase, endonuclease V. * To whom all correspondence should be addressed. 4071cell membrane is partially damaged by the action of the virus and then the structure is restored again. Thus, it seemed possible that macromolecules could be inserted into viable cells from the outside during the interaction between the cell membrane and HVJ. As an enzyme, T4 endonuclease V was used, which was isolated from Escherichia coli infected with bacteriophage T4 (10,11). This enzyme catalyzes the first step of excision repair in T4-infected cells (12). We tried to insert this enzyme into XP cells with help of HVJ, and this trial was successful. MATERIALS AND METHODSCells. Human fibroblasts derived from biopsy specimens of skin of normal subjects and XP patients were grown as monolayers in Eagle's minimum essential medium (MEM) supplemented with 10% fetal calf serum (Flow Lab.). Strain HNSF3 was derived from normal skin. Strains XP80S, XP1OOS, and XP6TOO were from Japanese XP patients and have been classified in complementation group A §. Strain CRL 1228, 1199, 1170, 1160, and 1259, belonging to the complementation groups A, B, C, D, and E, respectively, were obtain...

Unique DNA repair properties of a xeroderma pigmentosum revertant.

“…Experiments showing the importance of cyclobutane dimers in human cells usually involve exogenously irradiated plasmids or exogenously supplied microbial genes or gene products (9,24,25,34,35,37) (Table 3). These experiments may not accurately reflect how cells repair their own chromosomes with their endogenous enzyme systems.…”

Permeabilization of ultraviolet-irradiated Chinese hamster cells with polyethylene glycol and introduction of ultraviolet endonuclease from Micrococcus luteus.

“…3c, DNA from untreated cells incubated for 3 h had a size similar to that of DNA from cells examined immediately after irradiation (Fig. 3a) or after 24 h of incubation (data not shown). In cells treated with PEG plus extract, the DNA remained smaller than in untreated cells after 3 h of incubation.…”

“…Among the agents employed in permeabilization are hypertonic treatment (5), inactivated Sendai virus (23,24), and lysolecithin (14). This report describes the use of polyethylene glycol (PEG) to permeabilize Chinese hamster V-79 cells.…”

“…Cells from patients with the classic form of the hereditary disease xeroderma pigmentosum (XP) are defective in the excision repair of UV-induced pyrimidine dimers in DNA (7,20). However, UV-induced unscheduled DNA synthesis in these cells can be restored to the normal level, and cell survival can be significantly increased through the introduction of a UV-specific endonuclease (phage T4 endonuclease V) with inactivated Sendai virus as the permeabilizing agent (23,24). Isolated nuclei of XP cells incubated with the phage T4 endonuclease V also show an increase in repair replication (21).…”

“…In hairless mouse epidermis, 10% of the ESS are removed in 24 h after a dose in vivo, which corresponds to a dose in vitro of less than 2 J/m2 (13). Mouse 3T3 cells remove only 10% of the dimers induced by 5 was added, and the acid-insoluble precipitate was collected, washed twice with ethanol, and hydrolyzed in 95% formic acid.…”