Molecular analysis of enhanced replication of UV-damaged simian virus 40 DNA in UV-treated mammalian cells.

Irradiation of simian virus 40 (SV40)-infected cells with low fluences of UV light (20 to 60 J/m2, inducing one to three pyrimidine dimers per SV40 genome) causes a dramatic inhibition of viral DNA replication. However, treatment of cells with UV radiation (20 J/m2) before infection with SV40 virus enhances the replication of UV-damaged viral DNA. To investigate the mechanism of this enhancement of replication, we analyzed the kinetics of synthesis and interconversion of viral replicative intermediates synthesized after UV irradiation of SV40-infected cells that had been pretreated with UV radiation. This enhancement did not appear to be due to an expansion of the size of the pool of replicative intermediates after irradiation of pretreated infected cells; the kinetics of incorporation of labeled thymidine into replicative intermediates were very similar after irradiation of infected control and pretreated cells. The major products of replication of SV40 DNA after UV irradiation at the low UV fluences used here were form II molecules with single-stranded gaps (relaxed circular intermediates). There did not appear to be a change in the proportion of these molecules synthesized when cells were pretreated with UV radiation. Thus, it is unlikely that a substantial amount of DNA synthesis occurs past pyrimidine dimers without leaving gaps. This conclusion is supported by the observation that the proportion of newly synthesized SV40 form I molecules that contain pyrimidine dimers was not increased in pretreated cells. Pulse-chase experiments suggested that there is a more efficient conversion of replicative intermediates into form I molecules in pretreated cells. This could be due to more efficient gap filling in relaxed circular intermediate molecules or to the release of blocked replication forks. Alternatively, the enhanced replication observed here may be due to an increase in the excision repair capacity of the pretreated cells.     

Periodic changes of chromatin organization associated with rearrangement of repair patches accompany DNA excision repair of mammalian cells

We have used 8-methoxypsoralen to probe the chromatin structure of mammalian cells in situ while they repair pyrimidine dimers or bulky lesions in DNA. We observed that excision repair of these DNA lesions is accompanied by periodic alterations of chromatin organization. In parallel, fluctuations of the rates of repair patch synthesis accompanied these structural changes. Taking advantage of the accessibility of free DNA domains for psoralen intercalation, we have developed a technique to quantitatively isolate the micrococcal nuclease-sensitive, free DNA fraction of native bulk chromatin. We have determined the location of newly synthesized repair patches relative to free DNA domains as a function of repair time. Extensive rearrangements of repair patches from these domains into micrococcal nuclease-resistant DNA were observed. Our results indicate that periodic changes of chromatin organization associated with rearrangement of repair patches accompany the process of excision repair in mammalian cells.     

A nonuniform distribution of excision repair synthesis in nucleosome core DNA

We have investigated the distribution in nucleosome core DNA of nucleotides incorporated by excision repair synthesis occurring immediately after UV irradiation in human cells. We show that the differences previously observed for whole nuclei between the DNase I digestion profiles of repaired DNA (following its refolding into a nucleosome structure) and bulk DNA are obtained for isolated nucleosome core particles. Analysis of the differences obtained indicates that they could reflect a significant difference in the level of repair-incorporated nucleotides at different sites within the core DNA region. To test this possibility directly, we have used exonuclease III digestion of very homogeneous sized core particle DNA to "map" the distribution of repair synthesis in these regions. Our results indicate that in a significant fraction of the nucleosomes the 5' and 3' ends of the core DNA are markedly enhanced in repair-incorporated nucleotides relative to the central region of the core particle. A best fit analysis indicates that a good approximation of the data is obtained for a distribution where the core DNA is uniformly labeled from the 5' end to position 62 and from position 114 to the 3' end, with the 52-base central region being devoid of repair-incorporated nucleotides. This distribution accounts for all of the quantitative differences observed previously between repaired DNA and bulk DNA following the rapid phase of nucleosome rearrangement when it is assumed that linker DNA and the core DNA ends are repaired with equal efficiency and the nucleosome structure of newly repaired DNA is identical with that of bulk chromatin. Furthermore, the 52-base central region that is devoid of repair synthesis contains the lowest frequency cutting sites for DNase I in vitro, as well as the only "internal" locations where two (rather than one) histones interact with a 10-base segment of each DNA strand.     

The kinetics of thymine dimer excision in ultraviolet-irradiated human cells

We have investigated the kinetics of the loss of thymine dimers from the acid-insoluble fraction of several ultraviolet (UV)-irradiated cultured human cell lines. Our results show that UV fluences between 10 and 40 J/m2 produce an average of 21-85 x 10(5) thymine dimers per cell and an eventual maximal loss per cell of 12-20 x 10(5) thymine dimers. The time for half-maximal loss of dimers ranged from 12-22 h after UV irradiation. In contrast, the time for half-maximal repair synthesis of DNA measured by autoradiography was 4.5 h. This figure agrees well with reported half-maximal repair synthesis times, which range from 0.5 to 3.6 h based on our analysis. The discrepancy in the kinetics of the loss of thymine dimers from DNA and repair synthesis is discussed in terms of possible molecular mechanisms of thymine dimer excision in vivo and in terms of possible experimental artifacts.     

Preirradiation of host (monkey) cells mitigates the effects of UV upon simian virus 40 DNA replication

We are examining the effects of preirradiation of host (monkey) cells upon the replication of UV-damaged SV40. Control cells and cells preirradiated with low fluences (5 or 10 J/m2) of UV were infected with undamaged SV40, and the immediate effects of a subsequent irradiation were determined. UV inhibited total SV 40 DNA synthesis (incorporation of thymidine into viral DNA) in both preirradiated and control cells, but the extent of inhibition was less in the preirradiated cells. A test fluence of 60 J/m2 to SV40 replicating in preirradiated cells reduced synthesis only as much as a test fluence of 25 J/m2 in control cells. The fraction of recently replicated SV40 molecules that re-entered the replication pool and subsequently completed one round of replication in the first 2 h after UV was also decreased less in the preirradiated cells. Thus preirradiation of the host cell mitigates the immediate inhibitory effects of a subsequent UV exposure upon SV40 replication.     

Differential repair of 1-beta-D-arabinofuranosylcytosine-detectable sites in DNA of human fibroblasts exposed to ultraviolet light and 4-nitroquinoline 1-oxide

The extent of DNA excision repair was determined in dermal fibroblast strains from clinically normal and xeroderma pigmentosum (XP; complementation group A) human donors after single or combined exposures to 254-nm ultraviolet light and 4-nitroquinoline 1-oxide (4NQO). The repair was monitored by incubation of the treated cultures in the presence of 1-beta-D-arabinofuranosylcytosine (araC), a potent inhibitor of long-patch excision repair, followed by quantitation of araC-accumulated DNA single-strand breaks (representing repair events) by velocity sedimentation analysis in alkaline sucrose gradients. The amount of repair in normal fibroblast strains increased as a function of UV fluence and reached a plateau at 15 J/m2; strand breaks were not detected when these same cultures were irradiated with as much as 60 J/m2 UV and incubated in the absence of araC, implying that an initial (incision) step is rate-limiting in the repair of UV damage. In normal fibroblasts (i) the incidence of araC-detectable lesions removed during fixed intervals following exposure to 4NQO (4 microM; 30 min) was approximately 2.5 times greater than that seen following irradiation with repair-saturating fluences (greater than or equal to 15 J/m2) of UV-rays; and (ii) the amount of repair in cultures treated simultaneously with 4NQO (0.5-6 microM; 30 min) and a repair-saturating fluence of UV (20 J/m2) was found to approach the sum of that arising from exposure to each separately. The XP cells (XP12BE) exhibited a deficiency in the removal of araC-detectable DNA lesions following exposure to either of the carcinogens. Since araC is known to inhibit the repair of alkali-stable 4NQO-DNA adducts (i.e., lesions assumed to be removed by the UV-like excision pathway) but not that of alkali-labile sites (i.e., DNA lesions operated on by the X-ray-like repair pathway), our results strongly imply that the multistep excision-repair pathway operative on UV photoproducts in human fibroblasts differs from that responsible for removing alkali-stable (araC-detectable) 4NQO adducts by at least one step, presumably the rate-limiting incision reaction mediated by a lesion-recognizing endonuclease.     

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.     

Excision repair in permeable arrested human skin fibroblasts damaged by UV (254 nm) radiation: evidence that alpha- and beta-polymerases act sequentially at the repolymerisation step

We have characterised far-ultraviolet-radiation-induced DNA-repair synthesis in permeabilised arrested (non-dividing) primary human skin fibroblasts. Approximately half the maximum repair synthesis is seen after a UV fluence of 4.0 Jm-2 and little additional incorporation was observed at fluences above 20.0 Jm-2. UV-damaged permeable cells were treated with specific inhibitors of DNA polymerase alpha and beta, both alone and in combination. The degree of inhibition of repair incorporation by aphidicolin indicates that polymerase alpha is involved in the majority (85-90%) of repair synthesis after both high and low (less than 4.0 Jm-2) UV fluences. Dideoxythymidine triphosphate seems able to inhibit DNA-repair synthesis only when polymerase alpha is fully or almost fully functional, indicating that polymerase beta is unable to substitute in repair for an alpha polymerase blocked by aphidicolin. These data suggest that the two enzymes may act sequentially to complete repair patches rather than acting independently.     

Molecular and functional diversity of non-histone protein fraction NHCP1 from hamster Kirkman-Robbins hepatoma and liver

Non-histone protein fraction NHCP1 of micrococcal nuclease-sensitive and nuclease-resistant chromatin from Kirkman-Robbins hepatoma and hamster liver was studied by two-dimensional electrophoresis followed by Coomassie and silver staining and by microcomplement fixation technique in the presence of antibodies elicited against NHCP1 of both tissues. Apart from many common spots several tissue specific components associated with either nuclease-sensitive or nuclease-resistant chromatin were found. The presence of tissue specific components among NHCP1 from hepatoma and liver was confirmed by immunological analysis. It was stated that these components are exclusively localized in nuclease-resistant part of chromatin from neoplastic and normal tissues thus suggesting their structural function.     

Chromatin dynamics. Fast and slow modes of nucleosome movement revealed through psoralen binding and repair

Psoralen adducts, when formed in DNA at low frequencies that permit extensive survival of normal and repair-deficient cells, are found in both linker and core regions of nucleosomes, but are slightly enriched in the linker sites. The relative frequencies of adducts obtained with 5-methylisopsoralen and angelicin, which form only monoadducts, and 8-methoxypsoralen and trimethylpsoralen, which form monoadducts and cross-links, represent an enrichment in linker DNA that is approx. 2-3-fold higher per nucleotide than in core DNA. 5-Methylisopsoralen monoadducts, which are initially in linker DNA, become randomized during 12 h of growth. This suggests a slow lateral movement of nucleosomes with respect to DNA and implies that linker and core regions of DNA are not permanent assignments. Randomization of 5-methylisopsoralen adducts is independent of the synthesis of DNA, RNA, protein, or poly(ADP-ribose) and is also independent of DNA repair. Excision repair of these adducts, in contrast, causes rapid local changes in nucleosome conformation and an initial increase in staphylococcal nuclease sensitivity that reverts to the sensitivity of bulk chromatin in less than 1 h. Chromatin, therefore, can undergo at least two distinct dynamic changes under physiological conditions: a slow randomization of the nucleosomes with respect to DNA, and a rapid but transient local rearrangement to facilitate repair.     

Postreplication Repair of Ultraviolet Damage to DNA, DNA-Chain Elongation, and Effects of Metabolic Inhibitors in Mouse L Cells

Alkaline sucrose sedimentation studies of DNA from mouse L cells have demonstrated the following effects of several inhibitors of nucleic acid and protein synthesis on postreplication repair of ultraviolet (UV) damage to their DNA. The DNA newly synthesized by a 2 h [³H]thymidine (dThd) label following 254 nm UV irradiation of 20 J/m² is made in smaller segments of the number average mol wt (Mn) of ~10 × 10⁶ than the control of ~40 × 10⁶. The presence of caffeine at a concentration of 2 mM during the labeling of the irradiated cells reduces the Mn value to 5.8 × 10⁶, which is nearly comparable to, but somewhat larger than the expected distance between dimers in parental DNA. Afterwards, such an interrupted DNA made in the irradiated cells is completely repaired to the present maximum Mn value of 40 × 10⁶ in the consecutive 4 h chase in unlabeled dThd. The presence of the nucleic acid inhibitor, either 2 mM hydroxyurea, 50 μM arabinofuranosyl cytosine, 2 mM excess dThd or 5 μg/ml of actinomycin D (AMD) during 2- to 24-h chase periods after a 2 h postirradiation label prevents the repair to various extents, while 2 mM caffeine completely inhibits it. In the unirradiated cells, these agents except excess dThd and caffeine also interfere severely with normal elongation of nascent DNA made by a 3 min pulse label, but do not appreciably induce single chain breaks of either newly synthesized or parental DNA. The inhibition of the repair by AMD suggests that de novo elongation of DNA to close the gaps in new DNA made in the irradiated cells requires at least a template-dependent DNA polymerase. In contrast, 100 μg/ml of cycloheximide allows to complete the gap-filling repair, while it simply reduces the rates of chain growth for the repair and normal replication. Therefore, the similar sensitivity of gap-filling repair and normal replication towards the above inhibitors indicates that a preexisting DNA polymerizing system appears to be responsible and to play a common role without new protein synthesis, as far as the repair at early time after UV is concerned.     

Formation of DNA strand breaks in UV-irradiated human fibroblast preparations

The formation of DNA strand breaks was characterized in human fibroblasts prepared by several methods. In quiescent monolayer cultures of normal human fibroblasts (NHF), exposure to 254 nm radiation (UV) caused the rapid appearance of DNA strand breaks as monitored by alkaline elution analysis. Maximal levels of DNA breaks were seen 30 min after 10 J/m2; thereafter, strand breaks disappeared. Breakage soon after irradiation appeared to saturate at fluences above 10 J/m2. Xeroderma pigmentosum fibroblasts belonging to complementation group A (XPA) did not display this response which reflects operations of the nucleotidyl DNA excision repair pathway. When fibroblast strains were released from culture dishes by enzymatic digestion with trypsin or by scraping with a rubber policeman, UV-dependent DNA breakage displayed altered dose and time responses. Few breaks were detected in detached preparations of NHF after 10 J/m2 indicating inactivation of nucleotidyl DNA excision repair. The fluence response in detached fibroblasts was linear up to an incident fluence of 100 J/m2. Moreover, after 25 or 50 J/m2, strand breaks accumulated as a linear function of time for up to 2 h after irradiation. This UV-dependent and time-dependent incision activity was also observed in XPA monolayers and released-cell preparations. In permeable fibroblast preparations, DNA breaks accumulated in unirradiated cells that had been released with trypsin or by scraping. Permeabilization in situ saponin to open the plasma membrane produced a cell preparation that accumulated fewer UV-independent breaks. In saponin-permeabilized NHF that were irradiated with 10 J/m2, UV-dependent strand incision activity occurred at about 30% of the rate of incision seen in intact monolayer NHF. These results reveal at least 3 DNA strand incision activities in human fibroblast preparations of which only one reflects operation of the nucleotidyl DNA excision repair pathway.     

The endogenous nuclease sensitivity of repaired DNA in human fibroblasts

The limited DNA excision repair that occurs in the chromatin of UV-irradiated growth arrested cells isolated from a xeroderma pigmentosum (XP) complementation group C patient is clustered in localized regions. The repaired DNA was found to be more sensitive to nicking by endogenous nucleases than the bulk of the DNA. The extra-sensitivity does not change with increasing amounts of DNA damage or repair activity in the locally-repaired regions and is retained through a 24-h chase period. We suggest that these results are due to the occurrence of DNA repair limited to pre-existing, non-transient chromatin fractions that contain actively transcribed DNA. A similar extra-sensitivity of repaired DNA was not detected in cells of normal or XP complementation group A strains that exhibit either normal or limited repair located randomly throughout their genomes. The association between endogenous nuclease sensitivity and clustered repair probably defines a normal excision repair pathway that is specific for selected chromatin domains. The repair defect in XP-C strains may be one in pathways targeted for other endogenous nuclease-resistant domains.     

Enhanced DNA repair synthesis in hyperacetylated nucleosomes

We have investigated the level of "early" DNA repair synthesis in nucleosome subpopulations, varying in histone acetylation, from normal human fibroblasts treated with sodium butyrate. We find that repair synthesis occurring during the first 30 min after UV irradiation is significantly enhanced in hyperacetylated mononucleosomes. Nucleosomes with an average of 2.3 acetyl residues/H4 molecule contained approximately 1.8-fold more repair synthesis than nucleosomes with an average of 1.5 or 1.0 acetyl residues/H4 molecule. Fractionation of highly acetylated nucleosomes by two-dimensional gel electrophoresis yielded an additional 2.0-fold enrichment of repair synthesis in nucleosomes containing 2.7 acetyl residues/H4 molecule as compared to nucleosomes containing 1.9 acetyl residues/H4 molecule. This enhanced repair synthesis is associated primarily with nucleosome core regions and does not appear to result from increased UV damage in hyperacetylated chromatin. In addition, the distribution of repair synthesis within nucleosome core DNA from hyperacetylated chromatin is nonrandom, showing a bias toward the 5' end which is similar to that obtained for bulk (unfractionated) chromatin. These results provide strong evidence that enhanced repair occurs within nucleosome cores of hyperacetylated chromatin in butyrate-treated human cells. Finally, pulse-chase experiments demonstrate that the association of enhanced repair synthesis with hyperacetylated nucleosomes is transient, lasting only about 12 h after UV damage.     

Mechanisms of inhibition of DNA replication by ultraviolet light in normal human and xeroderma pigmentosum fibroblasts

The inhibition of DNA replication in ultraviolet-irradiated human fibroblasts was characterized by quantitative analysis of radiation-induced alterations in the steady-state distribution of sizes of pulse-labeled, nascent DNA. Low, noncytotoxic fluences (<1 J/m², producing less than one pyrimidine dimer per replicon) rapidly produced an inhibition of DNA synthesis in half-replicon-size replication intermediates without noticeably affecting synthesis in multi-repliconsize intermediates. With time, the inhibition produced by low fluences spread progressively to include multi-replicon-size intermediates. The results indicate that ultraviolet radiation inhibits the initiation of DNA synthesis in replicons. Higher (>1 J/m², producing more than one dimer per replicon) cytotoxic fluences inhibited DNA synthesis in operating replicons presumably because the elongation of nascent strands was blocked where pyrimidine dimers were present in template strands. Xeroderma pigmentosum fibroblasts with deficiencies in DNA excision repair exhibited an inhibition of replicon initiation after low radiation fluences. indicating the effect was not solely dependent upon operation of the nucleotidyl excision repair pathway. Owing to their inability to remove pyrimidine dimers ahead of DNA growing points, the repair-deficient cells also were more sensitive than normal cells to the ultraviolet-induced inhibition of chain elongation. Xeroderma pigmentosum cells belonging to the variant class were even more sensitive to inhibition of chain elongation than the repair-deficient strains despite their ability to remove pyrimidine dimers. This analysis suggests that normal and repair-deficient human fibroblasts either are able to rapidly bypass certain dimers or these dimers are not recognized by the chain elongation machinery.     

Similar distributions of repaired sites in chromatin of normal and xeroderma pigmentosum variant cells damaged by ultraviolet light

Excision repair of damage from ultraviolet light in both normal and xeroderma pigmentosum variant fibroblasts at early times after irradiation occurred preferentially in regions of DNA accessible to micrococcal nuclease digestion. These regions are predominantly the linker regions between nucleosomes in chromatin. The alterations reported at polymerization and ligation steps of excision repair in the variant are therefore not associated with changes in the relative distributions of repair sites in linker and core particle regions of DNA.     

Accommodation of pyrimidine dimers during replication of UV-damaged simian virus 40 DNA.

UV irradiation of simian virus 40-infected cells at fluences between 20 and 60 J/m2, which yield one to three pyrimidine dimers per simian virus 40 genome, leads to a fluence-dependent progressive decrease in simian virus 40 DNA replication as assayed by incorporation of [3H]deoxyribosylthymine into viral DNA. We used a variety of biochemical and biophysical techniques to show that this decrease is due to a block in the progression of replicative-intermediate molecules to completed form I molecules, with a concomitant decrease in the entry of molecules into the replicating pool. Despite this UV-induced inhibition of replication, some pyrimidine dimer-containing molecules become fully replicated after UV irradiation. The fraction of completed molecules containing dimers goes up with time such that by 3 h after a UV fluence of 40 J/m2, more than 50% of completed molecules contain pyrimidine dimers. We postulate that the cellular replication machinery can accommodate limited amounts of UV-induced damage and that the progressive decrease in simian virus 40 DNA synthesis after UV irradiation is due to the accumulation in the replication pool of blocked molecules containing levels of damage greater than that which can be tolerated.     

Dose-dependent increase in repair of 1-beta-D-arabinofuranosylcytosine-detectable DNA lesions in UV-treated xeroderma pigmentosum (group A) fibroblasts

The extent of DNA-excision repair was determined in human fibroblast strains from clinically normal and xeroderma pigmentosum complementation group A (XP-A) donors after irradiation with 254-nm ultraviolet (UV) light. Repair was monitored by the use of 1-beta-D-arabinofuranosylcytosine (araC), a potent inhibitor of DNA synthesis, and alkaline sucrose velocity sedimentation to quantitate DNA single-strand breaks. In this approach, the number of araC-accumulated breaks in post-UV incubated cultures becomes a measure of the efficiency of a particular strain to perform long-patch excision repair. The maximal rate of removal of araC-detectable DNA lesions equalled approximately 1.8 sites/10(8) dalton/h in the normal strains (GM38, GM43), while it was more than 10-fold lower in both XP-A strains (XP4LO, XP12BE) examined. In normal fibroblasts the number of lesions removed during the first 4 h after irradiation saturated at approximately 10 J/m2. In contrast, the residual amount of repair in the excision-deficient cells increased as a linear function of UV fluence over a range 5-120 J/m2. Thus we conclude that the repair of araC-detectable UV photoproducts in XP group A fibroblasts is limited by availability of damaged regions in the genome to repair complexes.     

The distribution of DNA excision-repair sites in human diploid fibroblasts following ultraviolet irradiation

Using the technique for separating DNA fragments containing excision-repair sites from total genomic DNA as described in the previous paper (Cohn, S. M., and Lieberman, M. W. (1984) J. Biol. Chem. 259, 12456-12462), we have developed a method for directly determining the distribution of excision-repair sites in the genome. DNA was prepared from confluent, diploid human fibroblasts which had been irradiated with ultraviolet light and incubated in the presence of 5-bromo-2'-deoxyuridine (BrdUrd), repaired fragments were isolated, and the dependence of the fraction of total DNA fragments containing excision-repair sites on DNA fragment length was determined by electrophoretic analysis. The observed dependence was compared to the relationship expected for a random distribution of repair sites. At 36 h following 3 J/m2 UV, the distribution of repair sites was indistinguishable from a random distribution; however, at doses of UV above 6 J/m2, the observed dependence indicated that the distribution of repair sites was nonrandom. A time course of the distribution of repair sites following 12 J/m2 UV was clearly nonrandom from 4 h after irradiation until at least 36 h following irradiation. By 72 h, however, the distribution had become random. In cells treated with hydroxyurea, a reduced number of excision-repair sites were present, but the distribution of repair sites was also nonrandom. Autoradiographic analysis of the amount of unscheduled DNA synthesis in individual nuclei suggested that the nonrandom distribution of repair sites did not result from variable extents of repair synthesis in different cell populations or from cell death.     

Measurement of the kinetics of DNA repair synthesis after uv irradiation using immunochemical staining of incorporated 5-bromo-2'-deoxyuridine and flow cytometry

The kinetics of unscheduled DNA synthesis in normal human fibroblasts was characterized by flow cytometry utilizing the immunofluorescent detection of 5-bromo-2'-deoxyuridine (BrdUrd) incorporated into cellular DNA during the repair process. Quiescent normal human fibroblasts were irradiated with ultraviolet light and incubated in the presence of BrdUrd during a postirradiation repair period. The amount of unscheduled DNA synthesis was then quantified in the quiescent cells by immunofluorescence staining using monoclonal antibodies against BrdUrd incorporated into the DNA. Significant amounts of unscheduled DNA synthesis were measured after doses as low as 0.1 J/m2 and for time periods as short as 15 min. The initial repair rate was found to be linear with time at all doses tested until repair neared completion. Interestingly, the initial repair rate was constant for doses over the range of 5 to 40 J/m2, whereas the time to completion of repair was dose dependent. These results suggest that above 5 J/m2 in normal human fibroblasts, the repair process is saturated but continues to function until all available regions are repaired. Using this methodology for measuring unscheduled DNA synthesis in combination with second and third flow markers, it is now possible to measure unscheduled DNA synthesis in heterogeneous mixtures of cells.