Lack of effect of hydroxyurea on base excision repair in mammalian cells

The effect of hydroxyurea on the initial steps of base excision repair has been examined in mammalian cells in 3 different proliferative states: i.e., quiescent cells, asynchronously growing cells undergoing multiple divisions prior to confluence; and synchronous cell populations undergoing the first cell cycle(s) after release from quiescence. Two parameters of the base excision repair pathway were examined: (1) The direct excision of 7-methylguanine from cellular DNA in the presence of increasing hydroxyurea concentrations was quantitated by high performance liquid chromatography; (2) the effects of hydroxyurea on the uracil DNA glycosylase were examined by quantitating the levels of this base excision repair enzyme in quiescent and proliferating cells. In quiescent cells, hydroxyurea at concentrations routinely used to quantitate DNA repair had no effect on the excision rates of 7-methylguanine examined over a span of 3 days; nor was there any effect on the specific activity of uracil DNA glycosylase in confluent cells. In asynchronously proliferating mammalian cells, identical hydroxyurea concentrations had no effect on the induction of the glycosylase. In synchronous growing cells HU had no effect on the temporal sequence of induction of uracil DNA glycosylase prior to DNA replication, nor on the extent of this induction. These results suggest that hydroxyurea at concentrations generally used to measure DNA repair has no effect on base excision repair.     

Cell cycle regulation of DNA repair in normal and repair deficient human cells

The regulation of nucleotide excision repair and base excision repair by normal and repair deficient human cells was determined. Synchronous cultures of WI-38 normal diploid fibroblasts and Xeroderma pigmentosum fibroblasts (complementation group D) (XP-D) were used to investigate whether DNA repair pathways were modulated during the cell cycle. Two criteria were used: (1) unscheduled DNA synthesis (UDS) in the presence of hydroxyurea (HU) after exposure to UV light or after exposure to N-acetoxy-acetylaminofluorene (N-AcO-AAF) to quantitate nucleotide excision repair or UDS after exposure to methylmethane sulfonate (MMS) to measure base excision repair; (2) repair replication into parental DNA in the absence of HU after exposure to UV light. Nucleotide excision repair after UV irradiation was induced in WI-38 fibroblasts during the cell cycle reaching a maximum in cultures exposed 14–15 h after cell stimulation. Similar results were observed after exposure to N-AcO-AAF. DNA repair was increased 2–4-fold after UV exposure and was increased 3-fold after N-AcO-AAF exposure. In either instance nucleotide excision repair was sequentially stimulated prior to the enhancement of base excision repair which was stimulated prior to the induction of DNA replication. In contrast XP-D failed to induce nucleotide excision repair after UV irradiation at any interval in the cell cycle. However, base excision repair and DNA replication were stimulated comparable to that enhancement observed in WI-38 cells. The distinctive induction of nucleotide excision repair and base excision repair prior to the onset of DNA replication suggests that separate DNA repair complexes may be formed during the eucaryotic cell cycle.     

Apurinic DNA endonuclease activities in repair-deficient human cell lines.

Several autosomal recessive diseases are associated with apparent DNA repair defects in cell culture. It seemed likely that a defect in excision repair reported for ataxia telangiectasia cells might reflect a lack of apurinic endonuclease activity. We report here normal levels of apurinic endonuclease activity in extracts of cell lines derived from patients with ataxia telangiectasia, xeroderma pigmentosum (complementation group D), Cockayne dwarfism, Fanconi anemia and Bloom syndrome.     

Human uracil DNA N-glycosidase: studies in normal and repair defective cultured fibroblasts.

Uracil DNA N-glycosidase, an enzyme which participates in the excision of uracil from DNA, was measured in extracts from fibroblasts lines cultured from normal subjects, from several subjects with the genetic disease xeroderma pigmentosum, and from a subject with ataxia telangiectasia. The cell lines representative of complementation groups A and D of xeroderma pigmentosum and of ataxia telangiectasia had roughly the same level of activity as did the normal cells. On the other hand, cells from two xeroderma pigmentosum variants (XP4BE and XP13BE) had roughly half the normal level of activity, and cells from the heterozygous mother of XP4BE had an intermediate level of activity. In spite of these quantitative differences, no systematic alterations in reaction characteristics, apparent Km for substrate, or purification characteristics were noted for enzyme from any of the lines. Thus a causal relationship, if any, between levels of activity and the disease symptoms is equivocal.     

Sensitization of rad mutants of Dictyostelium discoideum to ultraviolet light by postirradiation treatment with caffeine

The capacity of normal human cells to regulate DNA-repair pathways was examined. Synchronous populations of WI-38 human diploid fibroblasts were used to determine whether base-excision repair was increased as a function of the cell cycle. 2 parameters of the base-excision repair pathway were examined: (1) The induction of the DNA-repair enzyme uracil DNA glycosylase which functions in an initial step in base excision repair: (2) cell-mediated base-excision repair as measured by unscheduled DNA synthesis after exposure to sodium bisulfite or to methyl methanesulfonate. The glycosylase activity was increased 5-fold during cell proliferation; unscheduled DNA synthesis was enhanced 4- to 30-fold in a similar fashion. Equivalent results were observed where repair replication was quantitated using density-gradient analysis in the absence of hydroxyurea. The increase of the activity of the uracil DNA glycosylase and the enhancement of DNA repair occurred prior to the induction of DNA replication. Furthermore, at the maximal stimulation of DNA replication both glycosylase activity and DNA repair had substantially diminished. As the cells entered the second cell cycle, the glycosylase activity was again increased and then was again diminished. These results suggest that human cells actively modulate this DNA-repair pathway. The temporal stimulation of base-excision repair suggests the possibility that a DNA-repair complex may be formed prior to DNA replication to prescreen DNA and thus ensure the transfer of the correct genetic information to daughter cells.     

G2 phase repair of X-ray-induced chromosomal DNA damage in trichothiodystrophy cells

The repair of X-ray-induced DNA damage during G₂ cell-cycle phase has been examined in lines of skin fibroblasts from three patients with trichothiodystrophy (TTD), one with apparently normal and two with defective nucleotide excision repair (NER). These responses are compared with those of five lines from clinically normal controls, lines from xeroderma pigmentosum (XP), Cockayne syndrome (CS), Down syndrome (DS), and ataxia telangiectasia (AT) patients. Chromosomal DNA repair was measured as the chromatid aberration frequency (CAF) or total number of chromatid breaks and long gaps per 100 metaphase cells, determined 0.5–1.5 h after X-irradiation (53 rad). Chromatid breaks and gaps (as defined herein) represent unrepaired DNA strand breaks. Only one of the TTD lines, TTD 1BR, showed an abnormally high CAF. This line was shown subsequently to be of a different complementation group, representing a new nucleotide excision repair gene. An abnormally high CAF was also observed, as reported previously, in XP-C, AT and DS but not in CS skin fibroblasts. In addition, cell lines were examined for DNA incision activity by an indirect method in which chromatid aberrations were enumerated with or without ara-C, an inhibitor of repair synthesis, added after X-irradiation. All TTD lines had abnormally low incision activity.     

Requirement for ERCC-1 and ERCC-3 gene products in DNA excision repair in vitro. Complementation using rodent and human cell extracts.

Numerous rodent cell lines exist that have defects in nucleotide excision repair of DNA caused by alterations in genes that fall into 10 different complementation groups. The precise roles in the repair of these genes are unknown. We report here that extracts from Chinese hamster ovary cells of excision repair-defective complementation groups 1 and 3 are defective in DNA excision repair in a cell-free system. In vitro complementation can be achieved by mixing extracts from the two groups with one another. In addition, extracts from a human cell line representing xeroderma pigmentosum complementation group B could complement rodent complementation group 1 extracts, but not group 3 extracts. This is consistent with an identity of the ERCC-3 and xeroderma pigmentosum group B genes. Cellular evidence points toward a defect in the incision of damaged DNA in group 1 and 3 mutants. Since the ERCC-1 and ERCC-3 proteins are required for the in vitro reaction, it appears that both gene products are directly involved in the enzymatic incision of damaged DNA, or in preincision reactions. The experiments reported here provide the biochemical basis of an approach to analyze the function of these nucleotide excision repair proteins.     

Xeroderma pigmentosum group C protein interacts physically and functionally with thymine DNA glycosylase

The XPC-HR23B complex recognizes various helix-distorting lesions in DNA and initiates global genome nucleotide excision repair. Here we describe a novel functional interaction between XPC-HR23B and thymine DNA glycosylase (TDG), which initiates base excision repair (BER) of G/T mismatches generated by spontaneous deamination of 5-methylcytosine. XPC-HR23B stimulated TDG activity by promoting the release of TDG from abasic sites that result from the excision of mismatched T bases. In the presence of AP endonuclease (APE), XPC-HR23B had an additive effect on the enzymatic turnover of TDG without significantly inhibiting the subsequent action of APE. Our observations suggest that XPC-HR23B may participate in BER of G/T mismatches, thereby contributing to the suppression of spontaneous mutations that may be one of the contributory factors for the promotion of carcinogenesis in xeroderma pigmentosum genetic complementation group C patients.     

Conversion of replicative intermediates in human DNA-repair defective cells

We have examined the conversion of intermediates of DNA replication in normal human skin fibroblasts and fibroblasts isolated from patients with genetic diseases caused by putative DNA repair defects. Experiments were performed in non-transformed, unchallenged cells using alkaline sucrose sedimentation analysis to demonstrate precursor low molecular weight (LMW) DNA molecules which converted into high molecular weight (HMW) DNA with time. Analyses of conversion of replicative intermediates were conducted in cells from patients with ataxia telangiectasia (AT), Fanconi anemia (FA), Bloom syndrome (BS), Cockayne syndrome (CS) and xeroderma pigmentosum (XP). Our studies show that conversion of replicative intermediates occurs in all cell strains examined. However, XP cells (complementation groups A and E) show evidence of abnormalities in the conversion of LMW replicative intermediates, with the most dramatic alterations shown by cells from complementation group A.     

UV stimulation of DNA-mediated transformation of human cells.

Irradiation of dominant marker DNA with UV light (150 to 1,000 J/m2) was found to stimulate the transformation of human cells by this marker from two- to more than fourfold. This phenomenon is also displayed by xeroderma pigmentosum cells (complementation groups A and F), which are deficient in the excision repair of UV-induced pyrimidine dimers in the DNA. Also, exposure to UV of the transfected (xeroderma pigmentosum) cells enhanced the transfection efficiency. Removal of the pyrimidine dimers from the DNA by photoreactivating enzyme before transfection completely abolished the stimulatory effect, indicating that dimer lesions are mainly responsible for the observed enhancement. A similar stimulation of the transformation efficiency is exerted by 2-acetoxy-2-acetylaminofluorene modification of the DNA. No stimulation was found after damaging vector DNA by treatment with DNase or gamma rays. These findings suggest that lesions which are targets for the excision repair pathway induce the increase in transformation frequency. The stimulation was found to be independent of sequence homology between the irradiated DNA and the host chromosomal DNA. Therefore, the increase of the transformation frequency is not caused by a mechanism inducing homologous recombination between these two DNAs. UV treatment of DNA before transfection did not have a significant effect on the amount of DNA integrated into the xeroderma pigmentosum genome.     

Repair of bleomycin-damaged DNA by human fibroblasts

The ability of human fibroblasts to repair bleomycin-damaged DNA was examined in vivo. Repair of the specific lesions caused by bleomycin (BLM) was investigated in normal cell strains as well as those isolated from patients with apparent DNA repair defects. The diseases ataxia telangiectasia (AT), Bloom syndrome (BS), Cockayne syndrome (CS), Fanconi anemia (FA), and xeroderma pigmentosum (XP) were those selected for study. The method used for studying the repair of DNA after BLM exposure was alkaline sucrose gradient centrifugation. After exposure to BLM, a fall in the molecular weight of DNA was observed, and after drug removal the DNA reformed rapidly to high molecular weight. The fall in molecular weight upon exposure to BLM was observed in all cells examined with the exception of some XP strains. Prelabeled cells from some XP complementation groups were found to have a higher percentage of low molecular weight DNA on alkaline gradients than did normal cells. This prelabeled low molecular weight DNA disappeared upon exposure to BLM.     

α-N-Methylation of Damaged DNA-binding Protein 2 (DDB2) and Its Function in Nucleotide Excision Repair

DDB2 exhibits a high affinity toward UV-damaged DNA, and it is involved in the initial steps of global genome nucleotide excision repair. Mutations in the DDB2 gene cause the genetic complementation group E of xeroderma pigmentosum, an autosomal recessive disease manifested clinically by hypersensitivity to sunlight exposure and an increased predisposition to skin cancer. Here we found that, in human cells, the initiating methionine residue in DDB2 was removed and that the N-terminal alanine could be methylated on its α-amino group in human cells, with trimethylation being the major form. We also demonstrated that the α-N-methylation of DDB2 is catalyzed by the N-terminal RCC1 methyltransferase. In addition, a methylation-defective mutant of DDB2 displayed diminished nuclear localization and was recruited at a reduced efficiency to UV-induced cyclobutane pyrimidine dimer foci. Moreover, loss of this methylation conferred compromised ATM (ataxia telangiectasia mutated) activation, decreased efficiency in cyclobutane pyrimidine dimer repair, and elevated sensitivity of cells toward UV light exposure. Our study provides new knowledge about the posttranslational regulation of DDB2 and expands the biological functions of protein α-N-methylation to DNA repair.     

Interaction of nucleotide excision repair factors XPC-HR23B, XPA, and RPA with damaged DNA

The interaction of nucleotide excision repair factors--xeroderma pigmentosum complementation group C protein in complex with human homolog of yeast Rad23 protein (XPC-HR23B), replication protein A (RPA), and xeroderma pigmentosum complementation group A protein (XPA)--with 48-mer DNA duplexes imitating damaged DNA structures was investigated. All studied proteins demonstrated low specificity in binding to damaged DNA compared with undamaged DNA duplexes. RPA stimulates formation of XPC-HR23B complex with DNA, and when XPA and XPC-HR23B are simultaneously present in the reaction mixture a synergistic effect in binding of these proteins to DNA is observed. RPA crosslinks to DNA bearing photoreactive 5I-dUMP residue on one strand and fluorescein-substituted dUMP analog as a lesion in the opposite strand of DNA duplex and also stimulates cross-linking with XPC-HR23B. Therefore, RPA might be one of the main regulation factors at various stages of nucleotide excision repair. The data are in agreement with the cooperative binding model of nucleotide excision repair factors participating in pre-incision complex formation with DNA duplexes bearing damages.     

Cell-cycle defect of DNA repair in progeria skin fibroblasts

We examined the temporal regulation of DNA repair during synchronous cell proliferation in normal and progeroid human fibroblasts. Ultraviolet light-induced (254 nm, 20 J/m2) unscheduled DNA synthesis was measured at 4-h intervals after serum stimulation, for up to 32 h. Normal cells regulated DNA repair in a defined temporal sequence, showing a peak in the induction of DNA repair just before DNA synthesis. Progeroid skin fibroblasts failed to show an increase in nucleotide excision repair before scheduled DNA synthesis, but the background level of DNA repair was not significantly different from that in controls. Regulation of repair in progeroid human fibroblasts appeared similar, but not identical to that previously reported by Gupta and Sirover (1984b) for xeroderma pigmentosum complementation group C. Our results suggest that patients with Hutchinson-Gilford progeria may have a defect in DNA repair; the results offer nominal evidence that the average level of UV-induced DNA is decreased, and that individuals with this disease lack both the normal enhancement of DNA repair before scheduled DNA synthesis and the temporal control of DNA repair.     

Transformation of ultraviolet-irradiated human fibroblasts by simian virus 40 is enhanced by cellular DNA repair functions

Human fibroblasts irradiated with ultraviolet light were either tested for survival (colony formation) or infected with simian virus 40 and examined for transformation (foci formation). For normal cell cultures, the fractions of surviving colonies which were also transformed increased with increasing irradiation dose. In contrast, little increase in the transformation of ultraviolet-irradiated repair-deficient (xeroderma pigmentosum and xeroderma pigmentosum variant) cells was observed. Similar experiments with xeroderma pigmentosum variant cells treated with caffeine following irradiation indicated that, under these conditions, the deficient cells produced more transformants among the survivors of ultraviolet irradiation than did unirradiated cells. These results suggest (1) that DNA repair functions, not DNA damage per se, are required for enhanced viral transformation in normal cells; (2) that functions involved in excision repair and functions needed for replication of ultraviolet-damaged DNA appear necessary for this stimulation; and (3) that blocking DNA replication in ultraviolet-irradiated xeroderma pigmentosum variant cells by caffeine enhances viral transformation.     

Genetic recombination of herpes simplex virus, the role of the host cell and UV-irradiation of the virus

Summary Recombination frequencies for two sets of genetic markers of herpes simplex virus were determined in various host cells with and without ultraviolet irradiation of the virus. UV irradiation increased the recombination frequency in all the cell types studied in direct proportion to the unrepaired lethal damage. In human skin fibroblasts derived from a patient with xeroderma pigmentosum (XP) of complementation group A, a given dose of UV stimulated recombination more than that in fibroblasts from normal individuals. On the other hand, UV stimulation of HSV recombination was slightly less than normal in fibroblasts derived from a patient with a variant form XP and from an ataxia telangiectasia patient. Caffeine, an agent known to inhibit repair of UV damage, reduced recombination in most of the cell types studied but did not suppress the UV-induced increase in recombination. These findings suggest that for virus DNA with the same number of unrepaired UV-lesions, each of the tested cell types promoted HSV-recombination to an equivalent extent.     

Human xeroderma pigmentosum group G gene encodes a DNA endonuclease.

Because of defective nucleotide excision repair of ultraviolet damaged DNA, xeroderma pigmentosum (XP) patients suffer from a high incidence of skin cancers. Cell fusion studies have identified seven XP complementation groups, A to G. Previous studies have implicated the products of these seven XP genes in the recognition of ultraviolet-induced DNA damage and in incision of the damage-containing DNA strand. Here, we express the XPG-encoded protein in Sf9 insect cells and purify it to homogeneity. We demonstrate that XPG is a single-strand specific DNA endonuclease, thus identifying the catalytic role of the protein in nucleotide excision repair. We suggest that XPG nuclease acts on the single-stranded region created as a result of the combined action of the XPB helicase and XPD helicase at the DNA damage site.     

The post-incision steps of the DNA base excision repair pathway in Escherichia coli: studies with a closed circular DNA substrate containing a single U:G base pair.

The DNA base excision repair pathway is responsible for removal of oxidative and endogenous DNA base damage in both prokaryotes and eukaryotes. This pathway involves formation of an apurinic/apyrimidinic (AP) site in the DNA, which is further processed to restore the integrity of the DNA. In Escherichia coli it has been suggested that the major mode of repair involves replacement of a single nucleotide at the AP site, based on repair synthesis studies using oligonucleotide substrates containing a unique uracil base. The mechanism of the post-incision steps of the bacterial base excision repair pathway was examined using a DNA plasmid substrate containing a single U:G base pair. Repair synthesis carried out by repair-proficient ung, recJ and xon E.coli cell extracts was analyzed by restriction endonuclease cleavage of the DNA containing the uracil lesion. It was found that replacement of the uracil base was always accompanied by replacement of several nucleotides ( approximately 15) 3' of the uracil and this process was absolutely dependent on initial removal of the uracil base by the action of uracil-DNA glycosylase. In contrast to findings with oligonucleotide substrates, replacement of just a single nucleotide at the lesion site was not detected. These results suggest that repair patch length may be substrate dependent and a re-evaluation of the post-incision steps of base excision repair is suggested.     

Preferential repair of nuclear matrix associated DNA in xeroderma pigmentosum complementation group C

The distribution of ultraviolet-induced DNA repair patches in the genome of xeroderma pigmentosum cells of complementation group C was investigated by determining the molecular weight distribution of repair labeled DNA and prelabeled DNA in alkaline sucrose gradients after treatment with the dimerspecific endonuclease V of bacteriophage T4. The results were consistent with the data reported by Mansbridge and Hanawalt (1983) and suggest that DNA-repair synthesis in xeroderma pigmentosum cells of complementation group C occurs in localized regions of the genome. Analysis of the spatial distribution of ultraviolet-induced repair patches in DNA loops attached to the nuclear matrix revealed that in xeroderma pigmentosum cells of complementation group C repair patches are preferentially situated near the attachment sites of DNA loops at the nuclear matrix. In normal human fibroblasts we observed no enrichment of repair-labeled DNA at the nuclear matrix and repair patches appeared to be distributed randomly along the DNA loops. The enrichment of repair-labeled DNA at the nuclear matrix in xeroderma pigmentosum cells of complementation group C may indicate that the residual DNA-repair synthesis in these cells occurs preferentially in transcribing regions of the genome.