Rogerio Meneghini

Abstract— Mouse cells (3T3 line) and human fibroblasts are equally sensitive to UV light. At fluences of 2.0–2.5 J/m₂ mouse cells excise only 20% of the pyrimidine dimers as compared to 80% excised by human fibroblasts. This fluence allows 37% survival in both cases. Hence, mouse cells are more resistant to the same burden of unexcised dimers. The reason for this increased tolerance to dimers does not seem to be due to a recombinational mechanism, as judged by the fact that only ca. 5% of the dimers are transferred from parental to daughter strands. The transfer of dimers was measured by the Micrococcus luteus UV endonuclease assay, irradiating cells at Gi to avoid artifacts arising from introduction of dimers in nascent strands. The possibility of other mechanisms being involved in the process of tolerance to DNA lesions is discussed.     

Multiphoton near-infrared femtosecond laser pulse-induced DNA damage with and without the photosensitizer proflavine

The excitation of pBr322 supercoiled plasmid DNA with intense near-IR 810 nm fs laser pulses by a simultaneous multiphoton absorption mechanism results in single-strand breaks after treatment of the irradiated samples with Micrococcus luteus UV endonuclease. This enzyme cleaves DNA strands at sites of cyclobutane dimers that are formed by the simultaneous absorption of three (or more) 810 nm IR photons (pulse width approximately 140 fs, 76 MHz pulse repetition, average power output focused through 10x microscope objective is approximately 1.2 MW/cm2). Direct single-strand breaks (without treatment with M. luteus) were not observed under these conditions. However, in the presence of 6 microM of the intercalator proflavine (PF), both direct single- and double-strand breaks are observed under conditions where substantial fractions of undamaged supercoiled DNA molecules are still present. The fraction of direct double-strand breaks is 30 +/- 5% of all measurable strand cleavage events, is independent of dosage (up to 6.4 GJ/cm2) and is proportional to In, where I is the average power/area of the 810 nm fs laser pulses, and n = 3 +/- 1. The nicking of two DNA strands in the immediate vicinity of the excited PF molecules gives rise to this double-strand cleavage. In contrast, excitation of the same samples under low-power, single-photon absorption conditions (approximately 400-500 nm) gives rise predominantly to single-strand breaks, but some double-strand breaks are observed at the higher dosages. Thus, single-photon excitation with 400-500 nm light and multiphoton activation of PF by near-IR fs laser pulses produces different distributions of single- and double-strand breaks. These results suggest that DNA strand cleavage originates from unrelaxed, higher excited states when PF is excited by simultaneous IR multiphoton absorption processes.     

Gel electrophoresis method for quantitation of gamma ray induced single- and double-strand breaks in DNA irradiated in vitro

We describe a method based on gel electrophoresis for the quantitation of strand breaks in DNA and demonstrate its application to the measurement of single- and double-strand breaks formed by gamma-rays for DNA irradiated in vitro. For single-strand breaks, our data span the dose range from 0.1 to 1 Gy, while for double-strand breaks doses were from 3 to 15 Gy. In agreement with results obtained using other techniques, we find that the dose response function for single-strand breaks is linear while the dose response function for double-strand breaks is curved, indicating that it is the sum of both linear and quadratic components. We discuss factors that determine the sensitivity of the method and indicate approaches to make possible the quantitation of strand breaks in the DNA of cells irradiated with sublethal doses.     

PRODUCTION OF BREAKS IN SINGLE- AND DOUBLE-STRANDED FORMS OF BACTERIOPHAGE øx174 DNA BY PROFLAVINE AND LIGHT TREATMENT

Abstract— Irradiation at 440 + 360 nm and a fluence rate of 3.8 kJm^-2 min^-1, of both complexes previously formed between proflavine and either øX circular single-stranded (ss) DNA or øX supercoiled duplex (RFI)DNA, induces single-strand scissions in the two DNAs under consideration. Linear øXSS DNA molecules are detected by sedimentation through alkaline sucrose gradients. After treatment of the øXRFI DNA, however, the degree of degradation is the same whether it is measured under neutral or alkaline conditions, indicating that alkaline-labile bonds are not created; moreover, double-strand breaks can only be detected after accumulation of single-strand breaks. In addition to the amount of proflavine bound to the DNA and the duration of irradiation, the following factors are shown to influence the nicking activity of the treatment: (1) the DNA structure (the øXRFI DNA is much more sensitive than the øXss DNA); (2) the ionic strength of the medium during irradiation (a high value of 0.5 leads to a markedly increased efficiency); (3) the addition of cysteamine (this latter compound decreases the reaction rate) and (4) the irradiation wavelength (after irradiation at 440 nm alone, the reaction occurs at a reduced rate and is sensitive to NaN₃). The kinetics of the nicking reaction does not follow a single-hit curve showing that at least one primary lesion occurs prior to strand breakage. On the other hand, strand scission cannot be detected after irradiation of the proflavine-DNA complexes at the low fluence rate causing a decrease in the infectivity of both øXSS and øXRFI DNAs. Similarly. the sedimentation pattern of the DNA extracted from treated øx174 phages 99.9% inactivated, is identical to that of the control ss DNA, although more drastic treatments are susceptible to induce single strand breaks inside the phage head. Finally, the unknown lesion (s) that is biologically important does not prevent the treated DNAs from penetrating into the hostcells.     

WAVELENGTH DEPENDENCE OF THE FORMATION OF SINGLE-STRAND BREAKS AND BASE CHANGES IN DNA BY THE ULTRAVIOLET RADIATION ABOVE 150 nm

The wavelength dependence of the formation of two types of DNA damage, single-strand breaks and base changes, was investigated in the UV region from 150 nm to 254 nm using superhelical closed circular (form I) colicin El DNA with synchrotron radiation. Single-strand breaks were measured by agarose gel electrophoresis as a direct conversion of form I DNA to form II DNA (open circular). Base damages were defined as sensitive sites to a crude extract of endonuclease from Micrococcus luteus. They also were estimated using the same conversion, from form I to form II after the DNA was treated with endonuclease. The fluence-effect relationship could be fitted by a simple exponential function for both types of damage. Action spectra were constructed based on the reciprocal of the 37% fluence. The action spectrum for strand breaks increased rather monotonically over three decades from 254 nm to 150 nm in a logarithmic scale, while that for base damages showed a breaking point at 190 nm, being relatively flat above 190 nm. The characteristics of the action spectra are compared with the absorption spectra of the DNA and its main chain moiety calculated on the basis of data on calf thymus DNA and synthetic polynucleotides. Our main conclusions are (1) that the majority of single-strand breaks were induced by the absorption of photon in the sugar-phosphate group in the vacuum-UV region and (2) that the base changes were induced equally well by absorption in the vacuum-UV and in the far-UV region.     

DNA TURNOVER IN BUFFER-HELD ESCHERICHIA COLI AND ITS EFFECT ON REPAIR OF UV DAMAGE

Abstract— Continuous DNA degradation and resynthesis, without a net change in cellular DNA content, were observed in buffer-held, non-irradiated E. coli B/r. This constant DNA turnover probably involves most of the genome and reflects random sites of DNA repair due to the polA-dependent excision-resynthesis repair pathway. Under these non-growth conditions, it appears that at any given time there is a minimum of one repair site per 6.5 × 10⁶ daltons DNA, each of which is at least 160 nucleotides long.
While the amount of DNA degradation is not influenced by prior exposure to UV radiation, the synthetic activity decreases with increasing UV fluence. We suggest that when sites of DNA turnover occur opposite to cyclobutyl dipyrimidines in UV-irradiated cells, repair of the latter damage can be prevented. This implies that both beneficial and deleterious processes take place in irradiated buffer-held cells, and that cell survival depends on the delicate balance between DNA turnover and repair of UV-damage. Based on these findings, we propose a model to explain the limited repair observed during post-irradiation liquid-holding and to account for the large difference in cell survival between irradiation at low fluence rates (fluence-rate dependent recovery) and at high fluence rates followed by liquid-holding (liquid-holding recovery).     

SURVIVAL AND PYRIMIDINE DIMERS IN CULTURED FISH CELLS EXPOSED TO CONCURRENT SUN LAMP ULTRAVIOLET AND PHOTOREACTIVATING RADIATIONS

Abstract— Cultured fishcells(RBCF–1 line) were irradiated with filtered sun lamp ultraviolet (SL-UV; > 280 nm) together with or followed by illumination with daylight(DL) radiation (> 350 nm). The colony forming ability of the cells decreased with increasing fluence of SL-UV. Concurrent exposure of cells to SL-UV and DL, however, increased survival relative to exposure to SL-UV alone. The photoreactivable fraction reached 0.52 at22–25_‡C. By using a constant fluence modification factor of 86, the shape of dose-survival curve was found to be almost the same for 254 nm and SL-UV. In parallel with photoreactivation of cell survival, changes in the numbers of pyrimidine dimers in permeabilized cell DNA and in extracted total DNA were determined by measurements of endonuclease-sensitive sites (ESS). The yield of ESS in both DNA's increased almost linearly with increasing SL-UV fluence, although the yield in extracted DNA was about double of that in permeabilized cell DNA. The yield of ESS per unit fluence by 254 nm was about 70-fold greater than SL-UV. The fraction of cells inactivated per ESS was almost the same for 254 UV and SL-UV. In SL-UV-irradiated cells, the photoreactivable fractions in terms of ESS were _‡ 10% higher in extracted DNA than in the DNA of permeabilized cells and also were higher when DL was administered separately after SL-UV-irradiation. When irradiated cells were exposed to DL at 0_‡C, the photoreactivable fractions of both DNAs were appreciably less, indicating that the photoreactivation of ESS was enzymatic. These results support the suggestion that the mechanism for cell killing, mainly formation of pyrimidine dimers, by SL-UV is the same as that by 254 UV.     

INDUCTION OF DNA-PROTEIN CROSS-LINKS IN CHINESE HAMSTER CELLS BY THE PHOTODYNAMIC ACTION OF CHLOROALUMINUM PHTHALOCYANINE AND VISIBLE LIGHT

Abstract— Chloroaluminum phthalocyanine (CAPC) is an efficient photosensitizer for the inactivation of Chinese hamster V79 cells. In order to investigate possible molecular mechanisms in the photo-dynamic action of CAPC and visible light, the induction and repair rate of two classes of DNA lesions have been determined, i.e. DNA single-strand breaks and DNA-protein cross-links. In cells pretreated with 1 μ.M CAPC, a fluence of 12 kJ/m² of red light (>600 nm) kills approximately 50% of the cells and induces 3 to 3.5 Gy-equivalents of single-strand breaks. The repair of these breaks was slower than the repair of single-strand breaks induced by -irradiation. The photodynamic action of CAPC also induces a large number of DNA-protein cross-links which, in contrast to -radiation-induced DNA-protein cross-links, do not appear to be repaired during 4 h of post-treatment incubation in fresh medium. These studies suggest that DNA may be an important target for the cytotoxicity of CAPC + red light.     

Three-dimensional organization of block copolymers on "DNA-minimal" scaffolds

Here, we introduce a 3D-DNA construction method that assembles a minimum number of DNA strands in quantitative yield, to give a scaffold with a large number of single-stranded arms. This DNA frame is used as a core structure to organize other functional materials in 3D as the shell. We use the ring-opening metathesis polymerization (ROMP) to generate block copolymers that are covalently attached to DNA strands. Site-specific hybridization of these DNA-polymer chains on the single-stranded arms of the 3D-DNA scaffold gives efficient access to DNA-block copolymer cages. These biohybrid cages possess polymer chains that are programmably positioned in three dimensions on a DNA core and display increased nuclease resistance as compared to unfunctionalized DNA cages.     

Single-cell gel/comet assay applied to the analysis of UV radiation-induced DNA damage in Rhodomonas sp. (Cryptophyta).

The single-cell gel/comet assay is an electrophoretic technique used to detect single-strand breaks in DNA. Damage is assessed examining individual cells under an epifluorescent microscope. UV-induced DNA damage consists mostly of the formation of pyrimidine dimers; therefore, most of the damage cannot be detected using a standard comet assay. The enzyme T4 endonuclease V breaks DNA strands at sites of pyrimidine dimers. The main objective of this work is to evaluate the comet assay to detect UV-induced damage in DNA after an initial treatment of cells with T4 endonuclease V. This work was conducted on Rhodomonas sp. (Cryptophyta), a marine unicellular flagellate. Cells of Rhodomonas sp. were exposed to 12 h visible + ultraviolet-A + ultraviolet-B (VIS + UVA + UVB) and VIS (control), with and without T4 endonuclease V. Cells exposed to VIS + UVA + UVB showed approximately 200% more damage than control if these were treated with T4 endonuclease V. Rhodomonas sp. were exposed to 3, 6, 9 and 12 h of VIS, VIS + UVA and VIS + UVA + UVB. Damage induced by VIS + UVA + UVB as detected by the comet assay increased along with exposure time. However, damage caused by VIS and VIS + UVA remained relatively constant at all times. Results of this study indicate that the comet assay is more sensitive to UV radiation damage when used in conjunction with T4 endonuclease V. This modification of the comet assay can be used as an alternative technique to detect DNA damage in single cells caused by UV radiation.     

Double-strand hydrolysis of plasmid DNA by dicerium complexes at 37 degrees C.

Significant effort has been made to develop synthetic metal complexes that hydrolyze DNA. Here we report a new dicerium complex, Ce(2)(HXTA) (HXTA = 5-methyl-2-hydroxy-1,3-xylene-alpha,alpha-diamine-N,N,N',N'-tetraacetic acid), which can hydrolyze DNA at pH 8 and 37 degrees C. This complex hydrolyzes DNA restriction fragments to give products with high regioselectivity, affording >90% 5'-OPO(3) and 3'-OH ends, like the products of DNA hydrolyzing enzymes. Ce(2)(HXTA) also hydrolyzes Litmus 29 plasmid DNA to afford both nicked and linear DNA. Analysis of the relative amounts of supercoiled, nicked, and linear DNA present show that there is one double-strand cleavage per ten single-strand cleavages, indicating that the linear DNA formed cannot be the result of two random single-strand cleavage events. The kinetics of nicked and linear DNA formation are comparable, both being associated with apparent first-order rate constants of approximately 1 x 10(-)(4) s(-)(1) for complex concentrations of 10(-)(5)-10(-)(4) M. These observations suggest that similar factors affect the hydrolysis of the first and second DNA strands and that cleaving the phosphodiester bond is likely the rate determining step in both cases. This is the first detailed study of a metal complex shown to mimic DNA hydrolases in their capability to effect double-strand DNA hydrolysis regioselectively at the 3'-O-P bond.     

DNA LESIONS AND DNA DEGRADATION IN MOUSE LYMPHOMA L5178Y CELLS AFTER PHOTODYNAMIC TREATMENT SENSITIZED BY CHLOROALUMINUM PHTHALOCYANINE

Two closely related strains of mouse lymphoma L5178Y cells, LY-R and LY-S, have been found to differ in their sensitivity to the cytotoxic effects of photodynamic treatment (PDT) with chloroaluminum phthalocyanine (CAPC) and red light. Strain LY-R is more sensitive to photodynamic cell killing than strain LY-S. Differences in uptake of CAPC could not account for the differences in cytotoxic effects. There was no marked difference between the two strains in the induction of single-strand breaks (which includes frank single-strand breaks and alkali-labile lesions), but substantially more DNA-protein cross-links were formed in strain LY-R by CAPC and light. Repair of single-strand breaks proceeded with similar kinetics in both strains for the first 30 min post-irradiation, suggesting that these lesions are not responsible for the differential sensitivity of the two strains to the lethal effects of photodynamic treatment. Thereafter, alkaline elution revealed the presence of increasing DNA strand breakage in strain LY-R. DNA degradation, as measured by the conversion of prelabeled [14C] DNA to acid-soluble radioactivity, was more rapid and extensive in strain LY-R.     

RECOVERY OF SUBCHROMOSOMAL DNA SYNTHESIS IN SYNCHRONOUS V-79 CHINESE HAMSTER CELLS AFTER ULTRAVIOLET LIGHT EXPOSURE

Abstract— Previous work obtained from Chinese hamster V-79 cells indicated that, immediately following exposure, UV-induced lesions acted as blocks to elongation of nascent strands, but gradually lost that ability over a 10 h period after exposure to 10 J/m². The work reported herein attempted to examine possible cell cycle mediated alterations in the recovery of DNA synthesis. Kinetic incorporation of radiolabeled thymidine studies indicated that there may have been a more rapid recovery of DNA synthesis in cells irradiated in G₁ or G₂ vs cells irradiated in S phase. DNA fiber autoradiograms prepared from synchronous cells indicated that after irradiation in any phase of the cell cycle, the length of newly synthesized DNA was equal to control lengths 1 h after exposure to 5.0 J/m² (or 1 h after entering S phase for cells irradiated in G₁ or G₂). This observed recovery was not solely due to an excision process. No cell cycle mediated difference in the number of dimers induced or removed as a function of cell cycle position was observed. These results appear to be consistent with a continuum of effects, with initiation effects dominating the response at low fluences, gapped synthesis at intermediate fluences and elongation inhibition at high fluences. The fluences at which each event dominates may be cell-line specific.     

UV-INDUCED DNA DEGRADATION IN THE CYANOBACTERIUM SYNECHOCYSTIS PCC 6308

Ultraviolet radiation-induced DNA degradation has been demonstrated in the unicellular cyanobacterium Synechocystis PCC 6308. The extent of DNA degradation was greatly reduced by inhibition of DNA replication by preirradiation dark incubation and degradation was completely inhibited by exposure of irradiated cells to photoreactivating wavelengths. DNA degradation was not observed when irradiated cells were incubated in the presence of the excision repair inhibitors caffeine and acriflavin, suggesting that degradation is a manifestation of excision repair of pyrimidine dimers in Synechocystis . Increasing UV fluences resulted in an increase in the final extent of DNA degradation. However, at higher fluences degradation was inhibited, suggesting saturation of the excision repair system. Incubation of irradiated cells under conditions which inhibit protein synthesis greatly increased the extent of DNA degradation and the time over which it occurred. Exposure of cells to a sublethal fluence of UV greatly reduced the extent of DNA degradation produced by a challenge fluence administered after 24 h incubation providing evidence for inducible DNA repair activity in cyanobacteria.     

3-CARBETHOXYPSORALEN-DNA PHOTOLESIONS: IDENTIFICATION AND QUANTITATIVE DETECTION IN YEAST AND MAMMALIAN CELLS OF THE TWO cis-syn DIASTEREOISOMERS FORMED WITH THYMIDINE

Abstract A chemical method for the identification and the quantitative detection of psoralen DNA furan-side photoadducts formed in cells is presented. It is based on an enzymatic digestion of the purified DNA extracted from the treated cells and a further separation by high performance liquid chromatography of the modified nucleosides coupled to a highly sensitive fluorescence analysis and detection. Using this method, 3-carbethoxypsoralen- DNA photoadducts formed in yeast and mammalian cells have been identified and quantified. The predominant photoadducts induced have been identified as two cis-syn dThd(⁵₆^4'_5')3-CPs diastereoisomers. In Chinese Hamster V79 cells treated with 3-carbethoxypsoralen at 50 μM and irradiated at 365 nm with an incident dose of 24 kJ/m², the two monoadducts could be quantitatively assessed at levels as low as 1.3 and 0.7 per 10 000 base pairs.     

PHYSIOLOGICAL EFFECTS OF ULTRAVIOLET LIGHTON DICTYOSTELIUM DISCOIDEUM SPORE GERMINATION

Abstract— The spore germination in Dictyostelium discoideum consists of four stages: activation, postactivation lag, swelling and emergence. Ultraviolet irradiation (total fluence of 250 J/m²) of spores at any time prior to late spore swelling allows full swelling, but inhibits the emergence of myxamoebae. In the case of freshly activated spores, a UV exposure time of 30 s (total fluence of 50 J/m²) is sufficient to reduce emergence to about 6% when measured after 24 h of incubation. This same fluence results in about 10% viability as measured by plaque forming ability. Experiments utilizing 'fractionated exposures' result in the same percentage inhibition of emergence as that found for 'single exposures' provided the total fluence is equivalent. The higher fluences (250 J/m²) which completely prevent emergence, do not affect the endogenous oxygen uptake of spores during swelling. Ultraviolet light irradiated spores respond to the same activation and deactivation treatments as control unirradiated spores. Ultraviolet irradiation after late spore swelling allows emergence to occur in only a small fraction of the population. This fraction of cells which can emerge after UV treatment is said to have passed a 'competence point', which is believed to be the time when all the events necessary for emergence have been completed. Though the sites of UV inactivation in spores can only be postulated at present, it is apparent that the initial stages of germination (activation, postactivation lag and spore swelling) occur independently of the UV sensitive sites. The final stage of germination (emergence), however, is dependent on UV sensitive functions.     

FORMATION OF SINGLE AND DOUBLE STRAND BREAKS IN DNA ULTRAVIOLET IRRADIATED AT HIGH INTENSITY

The induction of single-strand breaks (SSB) by two quantum processes in DNA is well established. We now report that biphotonic processes result in double-strand breaks (DSB) as well. pUC19 and bacteriophage M13 RF DNA were irradiated using an excimer laser (248 nm) at intensities of 10(7), 10(9), 10(10) and 10(11) W/m2 and doses up to 30 kJ/m2. The proportion of DNA as supercoil, open circular, linear and short fragments was determined by gel electrophoresis. Linear molecules were noted at fluences where supercoiled DNA was still present. The random occurrence of independent SSB in proximity to each other on opposite strands (producing linear DNA) implies introduction of numerous SSB per molecule in the sample. If so, supercoiled DNA that has sustained no SSB should not be observed. A model accounting for the amounts of supercoiled, open circular, linear and shorter fragments of DNA due to SSB, DSB and Scissions (opposition of two independently occurring SSB producing an apparent DSB) was developed, our experimental data and those of others were fit to the model, and quantum yields determined for SSB and DSB formation at each intensity. Results showed that high intensity laser radiation caused an increase in the quantum yields for both SSB and DSB formation. The mechanism of DSB formation is unknown, and may be due to simultaneous cleavage of both strands in one biphotonic event or the biased introduction of an SSB opposite a preexisting SSB, requiring two biphotonic events.     

HOECHST 33258 DYE GENERATES DNA-PROTEIN CROSS-LINKS DURING ULTRAVIOLET LIGHT-INDUCED PHOTOLYSIS OF BROMODEOXYURIDINE IN REPLICATED AND REPAIRED DNA

Abstract— Substitution of bromodeoxyuridine for thymidine in the DNA of mammalian cells sensitizes them to a range of wavelengths of ultraviolet light. Cells are also sensitized to photochemical reactions involving dyes such as Hoechst 33258, which is used to produce differential staining of chromatids according to their bromodeoxyuridine content. Irradiation with 313 nm light of human and hamster cells containing bromodeoxyuridine in their DNA produced single-strand breaks, detectable by alkaline elution, but no DNA-protein cross-links. Irradiation with 360 nm light in the presence of Hoechst 33258 produced extensive DNA-protein cross-linkage as well as single-strand breaks. These cross-links were observed in DNA containing bromodeoxyuridine incorporated by either semiconservative or repair replication, and may provide a method for identification of proteins in close proximity to replication forks or repair sites. When the protein was removed with proteinase K, bromodeoxyuridine in repair patches after irradiation by doses of ultraviolet (254 nm) light as low as 0.26 J/m² could readily be detected. Hoechst 33258-mediated photolysis, therefore, provides a sensitive new technique for measuring repair replication after ultraviolet light irradiation.     

SINGLE-STRAND BREAKS INDUCED IN BACILLUS SUBTILIS DNA BY ULTRAVIOLET LIGHT: ACTION SPECTRUM and PROPERTIES

Abstract— The induction of single-strand breaks (alkali-labile bonds plus frank breaks) in the DNA of Bacillus subtilis irradiated in vivo by monochromatic UV light at wavelengths from 254 to 434 nm was measured. The spectrum consists of a major far-UV (below 320 nm) component and a minor near-UV shoulder. A mutant deficient in DNA polymerase I accumulates breaks caused by near-UV (above 320 nm) wavelengths faster than the wild-type strain proficient in polymerase I. Measurable breaks in extracted DNA are induced at a higher frequency than those induced in vivo. Anoxia, glycerol, and diazobicyclo (2.2.2.) octane inhibit break formation in extracted DNA. Alkali-labile bonds induced by 365-nm UV radiation are largely (78%) covalent bond chain breaks, the remainder consists of true alkali-labile bonds, probably apurinic and apyrimidinic sites.     

ACTION OF HYDROGEN PEROXIDE ON HUMAN FIBROBLAST IN CULTURE

Abstract— Human fibroblasts in culture lose the capacity of proliferating when exposed to hydrogen peroxide in the concentration range of 1 to 10 μ M . The toxicity of H₂O₂ to xeroderma pigmentosum cells (XP12RO). defective in excision repair of lesions produced by UV-irradiation, was about twice as high as to cells proficient in excision repair (VA13). This compound produces single-strand breaks in intracellular DNA but not in purified DNA. These breaks are in situ physical discontinuities rather than alkali-labile bonds, and their generation occurs at the same extent at 4°C and 37° indicating that they are not produced by an endonuclease. The results favor the hypothesis that H₂O₂ reacts in the cell producing a radical species which brings about the formation of DNA single-strand breaks. These breaks are effectively repaired by both XP12RO and VA13 fibroblasts. The possible reason for the lethality of H₂O₂ is discussed.