Radiofrequency (microwave) radiation exposure of mammalian cells during UV-induced DNA repair synthesis

The effect of continuous-wave (CW) and pulsed-wave (PW) radiofrequency radiation (RFR) in the microwave range on UV-induced DNA repair has been investigated in MRC-5 normal human diploid fibroblasts. RFR exposure at power densities of 1 (or 5) and 10 mW/cm2 gave a maximum specific absorption rate (SAR) (at 10 mW/cm2) of 0.39 +/- 0.15 W/kg for 350 MHz RFR, 4.5 +/- 3.0 W/kg for 850 MHz RFR, and 2.7 +/- 1.6 W/kg for 1.2 GHz RFR. RFR exposures for 1 to 3 h at 37 degrees C, in either continuous-wave or pulsed-wave modes, had no effect on the rate of repair replication label incorporated into preexisting UV-damaged DNA. RFR exposures (PW), with a constant medium temperature of 39 degrees C at 350 and 850 MHz during the repair period after UV damage, also had no effect. Assay for induction of repair synthesis by RFR exposure alone in non-UV irradiated cells was negative for the 350-, 850-, and 1200-MHz CW and PW RFR at 37 degrees C and the 350- and 850-MHz PW RFR at 39 degrees C. RFR does not induce DNA repair under these exposure conditions. In preliminary experiments--with the tissue culture medium maintained at 39 degrees C and RFR exposures (PW) at the frequencies of 350, 850, and 1200 MHz--no effect on incorporation of [3H]thymidine into DNA undergoing semiconservative synthesis was observed.     

Intranuclear localization of UV-induced DNA repair in human VA13 cells

We have investigated the intranuclear localization of DNA-repair synthesis in G1-phase VA13 human cells. Ultraviolet-irradiated cells were permitted to perform unscheduled DNA synthesis in 3H-thymidine (3H-TdR) and then extracted with nonionic detergent and 2 M NaCl to produce nucleoids in which residual nuclear matrix was surrounded by an extended halo of DNA loops. Autoradiographic analysis of these structures permitted discrimination of DNA repair between the matrix and halo regions. Repair label in nucleoids prepared from cells after exposure to fluences of 2.5-30 J/m2 exhibited a dose-dependent association with the nuclear matrix, which ranged from 80% after 2.5 J/m2 to 50% after 30 J/m2. These results support the view that DNA repair is a nuclear matrix-associated process. This conclusion is in agreement with our preliminary study (Harless et al., 1983) and the results of McCready and Cook (1984) but contrasts with that of Mullenders et al. (1983). Questions concerning the differing experimental designs and their potential effects on the localization of DNA repair are discussed. The implications of these results to previous attempts to isolate chromatin factors associated with DNA repair are also considered.     

Detection and repair of a UV-induced photosensitive lesion in the DNA of human cells

Irradiation with UV light results in damage to the DNA of human cells. The most numerous lesions are pyrimidine dimers; however, other lesions are known to occur and may contribute to the overall deleterious effect of UV irradiation. We have observed evidence of a UV-induced lesion other than pyrimidine dimers in the DNA of human cells by measuring DNA strand breaks induced by irradiating with 313-nm light following UV (254-nm) irradiation. These breaks, measured by alkaline sucrose sedimentation, increased linearly with the dose of UV light over the range tested (10-40 J/m2). The breaks cannot be photolytically induced 5 h after a UV dose of 20 J/m2 in normal cells; however, in xeroderma pigmentosum variant cells, the breaks are inducible for up to 24 h after UV irradiation. Xeroderma pigmentosum group A cells in the same 5-h period show an increase in the number of strand breaks seen with 313-nm light photolysis from about 2 to 4 breaks/10(9) dalton DNA. These breaks can then be induced for up to 24 h. These data suggest that, in normal cells, the lesion responsible for this effect is rapidly repaired or altered; whereas, in xeroderma pigmentosum variant cells it seems to remain unchanged. Some change apparently occurs in the DNA of xeroderma pigmentosum group A cells which results in an increase in photolability. These data indicate a deficiency in DNA repair of xeroderma pigmentosum variant cells as well as in xeroderma pigmentosum group A cells.     

Long-patch DNA repair synthesis during base excision repair in mammalian cells

The base excision repair (BER) process removes base damage such as oxidation, alkylation or abasic sites. Two BER sub-pathways have been characterized using in vitro methods, and have been classified according to the length of the repair patch as either 'short-patch' BER (one nucleotide) or 'long-patch' BER (LP-BER; more than one nucleotide). To investigate the occurrence of LP-BER in vivo, we developed an assay using a plasmid containing a single modified base in the transcribed strand of the enhanced green fluorescent protein (EGFP) gene and a stop codon, based on a single-nucleotide mismatch, at varying distances on the 3' side of the lesion. The reversion of the stop codon occurs after DNA repair synthesis and restores EGFP expression after transfection of mismatch-repair-deficient cells. Repair patches longer than one nucleotide were observed for 55-80% or 80-100% of the plasmids with a mean length of 2-6 or 6-12 nucleotides for 8-oxo-7,8-dihydroguanine or a synthetic abasic site, respectively. These data show the existence of LP-BER in vivo, and emphasize the effect of the type of BER substrate lesion on both the yield and the extent of the LP-BER sub-pathway.     

Reduced DNA repair in progeria cells and effects of gamma-ray irradiation on UV-induced unscheduled DNA synthesis in normal and progeria cells

A reduction in the amount of UV-induced unscheduled DNA synthesis (UDS), and reduced cell survival and host-cell reactivation against UV exposure in Hutchinson-Gilford progeria syndrome cell strains were shown. UV-induced UDS in 4 progeria cell strains was 33-50% of the normal level. A similar reduction in the UV-induced UDS in normal cells was caused by gamma-ray irradiation to the cells before UV irradiation. The dose of gamma-rays required to cause a reduction in UDS of normal cells to the level of progeria cells was 40 Gy and the reduction was reversible after 2 days. In progeria cells, gamma-ray irradiation further reduced UDS with a lower gamma-ray dose required than in normal cells, and the reduction was also reversible but with less relative recovery than in normal cells. The presence of a 'built-in' defect in progeria cells responsible for the reduced DNA-repair capacity was suggested, and such defect may share a common mechanism with the reduction of UV-induced UDS in normal cells caused by gamma-ray irradiation.     

Excision repair of mouse and human fibroblast cells, and a factor affecting the amount of UV-induced unscheduled DNA synthesis

Excision-repair ability and the amount of unscheduled DNA synthesis (UDS) after UV irradiation of fibroblast cells (in vitro passage 5) from C57BL mouse embryos were compared with those of human skin fibroblast cells. UDS in the mouse cells was approximately 75% of that in the human cells, although the disappearance of T4 endonuclease-V-susceptible sites and the accumulation of single-strand breaks in the mouse cell DNA indicated that the excision-repair capacity of the mouse cells was 20-35% of that in the human cells. This apparent discrepancy was ascribed to the difference in intracellular dTTP pool size, which was approximately twice as large in the human cells as in the mouse cells. UDS may not be suitable as a quantitative measure of excision repair when comparing the cells from different species.     

DNA polymerases and repair synthesis in NER in human cells

The late steps of nucleotide excision repair, following incisions to remove the damaged section of DNA, comprise repair synthesis and ligation. In vitro and in vivo studies have shown the size of the repaired patch to be about 30 nucleotides. In vitro studies implicated the replicative polymerases in repair synthesis, but recent in vivo data have shown that several DNA polymerases and ligases are involved in these steps in human cells.     

UV-induced DNA repair synthesis in cells of patients with different forms of xeroderma pigmentosum and of heterozygotes

UV-induced DNA repair synthesis, as measured by autoradiography as well as by isopycnic centrifugation methods, was studied in a large number of cell strains from patients with the classic form of xeroderma pigmentosum (XP) or the De Sanctis-Cacchione syndrome (DSC) and several of their heterozygous parents. On the basis of the kinetics of repair synthesis in the cultured skin fibroblasts we have recognized four distinct groups of XP patients: (1) classic XP patients with low residual repair capacities, (2) classic XP patients with intermediate, but dose-dependent, levels of repair synthesis relative to the normal level, (3) patients, diagnosed as having classic XP, with a normal or only slightly reduced repair capacity and (4) DSC patients with a complete deficiency of repair synthesis. Complementation studies reported elsewhere have shown that different mutations are responsible for the detect in at least three of these groups. Cell strains of each of the four XP types were able to rejoin single-strand DNA breaks induced by X-rays. Most of the cell strains derived from heterozygotes showed normal repair activities. However, in the parents some of the of DSC-patients a significant reduction of the level of repair synthesis was found.     

UV-induced immobilization of DNA repair protein XPA in different human cell line

XPA repair protein is absolutely needed for nucleotide excision repair (NER). It preferentially binds UV-irradiated DNA in vitro and possibly takes place in the recognition of pyrimidine dimers, the main type of UV-lesions in DNA. Using immunofluorescent microscopy and immunoblotting technique we have found that XPA protein is fully extractable by Triton X-100 solution from non-irradiated normal human fibroblasts, but after UV-irradiation its extractability decreases in UV-dose dependent manner. UV-induced XPA-immobilization was observed in human cell lines with different types of repair defects, but XPA-extractability from unirradiated cells of these lines was significantly lower in comparison with normal fibroblasts. These data do not permit to make conclusion concerning the distinct connection of this phenomenon with different pathways of NER. Histone deacetylase inhibitor, sodium butyrate, did not change the level of extractability in unirradiated and UV-irradiated normal human cells and CHO cells, defective in global genome repair, that indicated the independence of XPA-immobilization from the level of histone acetylation. It was established with the help of confocal microscopy that XPA-foci in detergent-treated UV-irradiated cell were partially colocalized with the focal sites of PCNA, an auxiliary protein of DNA polymerases delta and epsilon. It may mean that a part of detergent-resistant XPA foci correspond to DNA repair synthesis sites, but the major part of immobilized XPA reflects the early step of repair proteins assembly formation needed for the repair of the lesions.     

Suppression of UV-induced apoptosis by the human DNA repair protein XPG

The severe xeroderma pigmentosum/Cockayne syndrome (XP/CS) syndrome is caused by mutations in the XPB, XPD and XPG genes that encode the helicase subunits of TFIIH and the 3' endonuclease of nucleotide excision repair (NER). Because XPB and XPD have been implicated in p53-mediated apoptosis, we examined the possible involvement of XPG in this process. After ultraviolet light (UV) irradiation, primary fibroblasts of XP complementation group G (XP-G) individuals with CS enter apoptosis more readily than other NER-deficient cells, but this is unlinked to unrepaired damage. These XP-G/CS cells accumulate p53 post-UV but they fail to accumulate the 90/92 kDa isoforms of Mdm2 and their cellular distribution of Mdm2 is impaired. Apoptosis levels revert to wild type, Mdm2 90/92 kDa isoforms accumulate, and Mdm2 regains its normal post-UV nuclear location in transduced XP-G/CS cells expressing wild-type XPG, but not an XPG catalytic site mutant. These results suggest that XPG suppresses UV-induced apoptosis and that this suppression, most simply, requires its endonuclease function.     

Organization of UV-induced,repair replicated DNA in the cellular genome of cultured human and mouse cells

The distribution of UV-induced repair-replicated DNA patches among reiterated and unique murine and human DNA has been studied by molecular reassociation. DNA-DNA renaturation was employed to fractionate labeled repair-replicated and normal cellular DNA sequences according to their reiteration frequencies. Results indicate that repair replicated DNA patches are distributed uniformly within highly repeated, moderately repeated and single copy DNA sequences. This could be due to the random localization of UV-induced lesions and repairs in the cultured murine and human cells.     

Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells

The eukaryotic sliding DNA clamp, proliferating cell nuclear antigen (PCNA), is essential for DNA replication and repair synthesis. In order to load the ring-shaped, homotrimeric PCNA onto the DNA double helix, the ATPase activity of the replication factor C (RFC) clamp loader complex is required. Although the recruitment of PCNA by RFC to DNA replication sites has well been documented, our understanding of its recruitment during DNA repair synthesis is limited. In this study, we analyzed the accumulation of endogenous and fluorescent-tagged proteins for DNA repair synthesis at the sites of DNA damage produced locally by UVA-laser micro-irradiation in HeLa cells. Accumulation kinetics and in vitro pull-down assays of the large subunit of RFC (RFC140) revealed that there are two distinct modes of recruitment of RFC to DNA damage, a simultaneous accumulation of RFC140 and PCNA caused by interaction between PCNA and the extreme N-terminus of RFC140 and a much faster accumulation of RFC140 than PCNA at the damaged site. Furthermore, RFC140 knock-down experiments showed that PCNA can accumulate at DNA damage independently of RFC. These results suggest that immediate accumulation of RFC and PCNA at DNA damage is only partly interdependent.     

Radioadaptive enhancement of the repair of UV-induced postreplication gaps in Escherichia coli cells deficient in DNA repair

In UV-irradiated E. coli WP2 uvrA, deficient in excision repair of DNA with pyrimidine dimers, gamma-irradiation in low doses (radioadaptation) before UV-irradiation leads to the intensification of postreplication repair of DNA. This process in WP2 uvrA polA and uvrA lexA mutants is less than in WP2 uvrA cells, but in WP2 uvrA recA both postreplication repair and its radioadaptive intensification are absent. In E. coli AB1157 excising pyrimidine dimers the radioadaptive intensification of postreplication repair of DNA is expressed almost to the same extent as in WP2 uvrA. In GW2100 umuC mutant, deficient in DNA polymerase V, postreplication repair of DNA is expressed, but its radioadaptive intensification is absent, while in AB2463 recA13 both postreplication repair of DNA and radioadaptive intensification of postreplication repair of DNA are absent. The above data suggest that DNA polymerase I and LexA protein are needed for radioadaptive intensification of postreplication repair of DNA in uvrA strain, and DNA polymerase V is needed for radioadaptive intensification in E. coli AB1157, and that RecA protein is required for postreplication repair and radioadaptive intensification of postreplication repair of DNA.     

Requirement for functional DNA polymerase eta in genome-wide repair of UV-induced DNA damage during S phase

The autosomal recessive disorder Xeroderma pigmentosum-variant (XPV) is characterized (i) at the cellular level by dramatic hypermutability and defective recovery of DNA synthesis following UV exposure, and (ii) clinically by abnormal sunlight sensitivity and remarkable predisposition to skin cancer. These phenotypes are clearly attributable to germline mutations in POLH, encoding DNA polymerase eta (polη) normally required for accurate translesion DNA synthesis (TLS) past UV-induced cyclobutane pyrimidine dimers. Here we demonstrate that patient-derived XPV-skin fibroblasts exposed to 15 J/m² of UV also exhibit (in addition to abnormal TLS) a significant defect in global-genomic nucleotide excision repair (GG-NER) exclusively during S phase. This cell cycle-specific GG-NER defect can be complemented by ectopic expression of wild-type polη, but not of polη variants deficient in either nuclear relocalization or PCNA interaction. We highlight a previous study from our laboratory demonstrating that UV-exposed, ATR-deficient Seckel syndrome fibroblasts, like XPV fibroblasts, manifest strong attenuation of GG-NER uniquely in S phase populations. We now present further evidence suggesting that deficient S phase repair can be rescued in both XPV- and Seckel syndrome-cells if the formation of blocked replication forks post-UV is either prevented or substantially reduced, i.e., following, respectively, pharmacological inhibition of DNA synthesis prior to UV irradiation, or exposure to a relatively low UV dose (5 J/m²). Our findings in cultured cells permit speculation that abrogation of GG-NER during S phase might partially contribute (in a synergistic manner with defective, atypically error-prone TLS) to the extreme state of UV-hypermutability leading to accelerated skin cancer development in XPV patients. Moreover, based on the overall data, we postulate that loss of either functional polη or -ATR engenders abnormal persistence of stalled replication forks at UV-adducted sites in DNA which, in turn, can actively and/or passively trigger GG-NER inhibition.     

Telomeric fusion of the chromosomes in cells with micronuclei during the subsequent incorporation into the DNA of thymidine halogenated analogs

As was shown elsewhere among halogenated analogs of thymidine there exist strong (5-chlorodeoxyuridine, 5-ChldU) and weak (5-iododeoxyuridine, 5-IdU) inducers of dicentric chromosomes in cells with micronuclei. Since from colcemid administration to the moment of fixation two cycles of replication are passed, a study was made of the pattern of induction of dicentric chromosomes under subsequent administration of 5-ChldU and 5-IdU, and the other way round. It was shown that the pattern of induction of dicentric chromosomes was strong or weak, and depended on the fact which analog was administered in the first S-period.     

Reversal of the changes in DNA and chromosome structure which follow the inhibition of UV-induced repair in human cells

The inhibition of UV-induced repair DNA synthesis by hydroxyurea, deoxyadenosine or 1-β-D-arabinofuranosylcytosine is reflected in changes in chromosome appearance (decondensation seen in fixed preparations) and DNA structure (accumulation of unwinding points or single-strand gaps). These changes are rapidly reversed if the inhibition is neutralised by addition of precursors of DNA synthesis. The $\text{in vivo}$ organisation of chromosomes apparently survives the prolonged presence of substantial DNA breakage.     

In situ enzymology of DNA replication and ultraviolet-induced DNA repair synthesis in permeable human cells

Using permeable diploid human fibroblasts, we have studied the deoxyribonucleoside triphosphate concentration dependences of ultraviolet- (UV-) induced DNA repair synthesis and semiconservative DNA replication. In both cell types (AG1518 and IMR-90) examined, the apparent Km values for dCTP, dGTP, and dTTP for DNA replication were between 1.2 and 2.9 microM. For UV-induced DNA repair synthesis, the apparent Km values were substantially lower, ranging from 0.11 to 0.44 microM for AG1518 cells and from 0.06 to 0.24 microM for IMR-90 cells. Control experiments established that these values were not significantly influenced by nucleotide degradation during the permeable cell incubations or by the presence of residual endogenous nucleotides within the permeable cells. Recent data implicate DNA polymerase delta in UV-induced repair synthesis and suggest that DNA polymerases alpha and delta are both involved in semiconservative replication. We measured Km values for dGTP and dTTP for polymerases alpha and delta, for comparison with the values for replication and repair synthesis. Km values for polymerase alpha were 2.0 microM for dGTP and 5.0 microM for dTTP. For polymerase delta, the Km values were 2.0 microM for dGTP and 3.5 microM for dTTP. The deoxyribonucleotide Km values for DNA polymerase delta are much greater than the Km values for UV-induced repair synthesis, suggesting that when polymerase delta functions in DNA repair, its characteristics are altered substantially either by association with accessory proteins or by direct posttranslational modification. In contrast, the deoxyribonucleotide binding characteristics of the DNA replication machinery differ little from those of the isolated DNA polymerases.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA replication and UV-induced DNA repair synthesis in human fibroblasts are much less sensitive than DNA polymerase alpha to inhibition by butylphenyl-deoxyguanosine triphosphate.

In mammalian cells, both semiconservative DNA replication and the DNA repair patch synthesis induced by high doses of ultraviolet radiation are known to be inhibited by aphidicolin, indicating the involvement in these processes of one or both of the aphidicolin-sensitive DNA polymerases, alpha and/or delta. In this paper, N2-(p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate, a strong inhibitor of polymerase alpha and a weak inhibitor of polymerase delta, is used to further characterize the DNA polymerase(s) involved in these two forms of nuclear DNA synthesis. In permeable human fibroblasts, DNA replication and ultraviolet-induced DNA repair synthesis are more resistant to the inhibitor than DNA polymerase alpha by factors of approximately 500 and 3000, respectively. These findings are most consistent with the involvement of DNA polymerase delta in these processes.