DNA repair and replication in human fibroblasts treated with (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

DNA repair and replication were examined in diploid human fibroblasts after treatment with (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I). Unscheduled DNA synthesis exhibited a linear response to BPDE-I concentrations up to 1.5 μM and a saturation plateau after higher concentrations. Maximal unscheduled DNA synthesis was observed in the first hour after treatment with synthesis diminishing progressively thereafter. Half-maximal unscheduled DNA synthesis was seen within 4–6 h after treatment with 0.7 μM BPDE-I. DNA replication was inhibited by BPDE-I in a dose- and time-dependent fashion. The mechanisms of this inhibition were characterized by velocity sedimentation of pulse-labeled nascent DNA in alkaline sucrose gradients. Very low concentrations of BPDE-I (0.03 and 0.07 μM) were found to inhibit replicon initiation by up to 50% within 30–60 min after treatment. Recovery of initiation following these low concentrations was evident within 3 h after treatment. Higher concentrations of carcinogen inhibited DNA synthesis in active replicons. This effect was manifested by a reduction in incorporation of precursor into replication intermediates of greater than 1·10⁷ Da with the concurrent production of abnormally small nascent DNA. When viewed 45 min after treatment with 0.17 μM BPDE-I the combination of these two effects partially masked the inhibition of replicon initiation. However, even after treatment with 0.33 μM BPDE-I an effect on initiation was evident. These results reveal a pattern of response to BPDE-I that is quite similar to that produced by 254 nm radiation.     

Effect of cytosine arabinoside on replicon initiation in human lymphoblasts

Cytosine arabinoside inhibited DNA synthesis in human lymphoblasts by inhibiting the initiation of DNA replication units. This effect was observed by a decrease in the incorporation of (³H) thymidine into low molecular weight DNA analyzed by velocity sedimentation in alkaline sucrose gradients. In contrast, there was no detectable effect on chain elongation and joining of those molecules that initiated replication before addition of the drug. These data indicate that cytosine arabinoside acts preferentially at the level of initiation of DNA replication rather than chain elongation.     

On the mode of action of 5-diazouracil on bacterial cell division

Cell division by strains ofEscherichia coli andSalmonella typhimurium is inhibited by 5-diazouracil (5-DU). Division recovers in the presence of the inhibitor after a period which is temperature-dependent. Recovery is probably due to breakdown of 5-DU and the rate of this breakdown is apparently increased at alkaline pH. Growth with 5-DU caused only a slight reduction in the rate of murein synthesis and no alteration in the properties or composition of membranes ofS. typhimurium. The agent caused chaining inStreptococcus fecalis and inhibition of the penicillin-induced lysis ofS. typhimurium. These effects may have been due to direct inhibition of lysin activity but an indirect effect seems more likely. The most marked effect of 5-DU onS. typhimurium was to cause a transient inhibition of DNA synthesis. Since 5-DU did not stop uncoupled cell division (i.e. division occurring independently of DNA replication) and sincelon^? strains were more sensitive to 5-DU thanlon⁺ strains, it was concluded that 5-DU acts on cell division via an inhibitory effect on DNA replication.     

Effects of inhibition of protein synthesis on DNA replication in cultured mammalian cells

We have investigated the effects of inhibiting protein synthesis on the overall rate of DNA synthesis and on the rate of replication fork movement in mammalian cells. In order to test the validity of using [³H]thymidine incorporation as a measure of the overall rate of DNA synthesis during inhibition of protein synthesis, we have directly measured the size and specific radioactivity of the cells' [³H]dTTP pool. In three different mammalian cell lines (mouse L, Chinese hamster ovary, and HeLa) nearly complete inhibition of protein synthesis has little effect on pool size (±26%) and even less effect on its specific radioactivity (±11%). Thus [³H]thymidine incorporation can be used to measure accurately changes in rate of DNA synthesis resulting from inhibition of protein synthesis.Using the assay of [³H]thymidine incorporation to measure rate of DNA synthesis, and the assay of [¹⁴C]leucine or [¹⁴C]valine incorporation to measure rate of protein synthesis, we have found that eight different methods of inhibiting protein synthesis (cycloheximide, puromycin, emetine, pactamycin, 2,4-dinitrophenol, the amino acid analogs canavanine and 5-methyl tryptophan, and a temperature-sensitive leucyl-transfer tRNA synthetase) all cause reduction in rate of DNA synthesis in mouse L, Chinese hamster ovary, or HeLa cells within two hours to a fairly constant plateau level which is approximately the same as the inhibited rate of protein synthesis.We have used DNA fiber autoradiography to measure accurately the rate of replication fork movement. The rate of movement is reduced at every replication fork within 15 minutes after inhibiting protein synthesis. For the first 30 to 60 minutes after inhibiting protein synthesis, the decline in rate of fork movement (measured by fiber autoradiography) satisfactorily accounts for the decline in rate of DNA synthesis (measured by [³H]thymidine incorporation). At longer times after inhibiting protein synthesis, inhibition of fork movement rate does not entirely account for inhibition of overall DNA synthesis. Indirect measurements by us and direct measurements suggest that the additional inhibition is the result of decline in the frequency of initiation of new replicons.     

Antagonistic effect of magnesium chloride on the nickel chloride–induced inhibition of DNA replication in chinese hamster ovary cells

The degree of inhibition of semiconservative DNA replication induced by nickel chloride (NiCl₂) was analyzed by radiolabeled-thymidine incorporation alone or with cesium chloride (CsCl) density gradient centrifugation. The onset and duration of this Ni²⁺-induced inhibition was time- and concentration- dependent, but the degree of inhibition was not. A maximal reduction in the rate of DNA synthesis was observed within the first hour of treatment with 2.5 mM NiCl₂, which was the highest noncytotoxic concentration utilized. After six hours, 500 μM and 1 mM as well as 2.5 mM NiCl₂ all produced the same 50% to 60% reduction in [³H]-thymidine incorporation into DNA. The inhibitory effect of nickel ions on DNA synthesis was reversible. The rate of DNA synthesis following a 500 μM or 1 mM NiCl₂ treatment began to increase after washout of nickel, but a six-hour exposure of cells to 2.5 mM NiCl₂ produced a sustained 50% to 60% suppression of DNA synthetic activity for at least 36 hours. At all concentrations of NiCl₂ used in this study, some inhibition of DNA synthesis persisted for at least 48 hours, but by 72 hours after treatment, the rate of [³H]-thymidine incorporation was actually 10% above the control. Examination of autoradiographic slides of cells treated with 2.5 mM NiCl₂ for six hours demonstrated a 60% reduction of silver grains, but there was no preferential reduction in the quantity of grains in the nucleolus or any other region. Cesium chloride density gradient analysis of the replication of nucleolar DNA in cells treated with 2.5 mM nickel supported the autoradiographic findings. The inhibitory effect of NiCl₂ on DNA replication was prevented by the addition of magnesium chloride (MgCl₂) to cells maintained in a simple salts/glucose medium (SGM). This effect did not appear to be due to an antagonism of the cellular uptake of nickel by Mg²⁺, since the maximally effective dose of Mg²⁺ reduced ⁶³Ni²⁺ uptake by no more than 25% while the inhibition of replication was completely reversed.     

Chloramphenicol effects on DNA replication in UV-damaged bacteria

Increasing UV-doses to cultures of Escherichia coli strain B/r decreased progressively the amount of DNA which was formed in the presence of chloramphenicol (160 μg/ml) from the amount formed in unirradiated control cultures in chloramphenicol-containing medium. This is attributed to the progressive inactivation of active sites of DNA replication by UV. In order to form DNA the bacteria must then replicate from the chromosomal fixed origin, an activity which requires protein synthesis and thus cannot occur in the presence of chloramphenicol. Such damage was shown to be subject to photoreactivation after lower UV-doses and thus is the pyrimidine dimer. After higher doses non-photoreversible lesions began to accumulate so that all such damage became non-photoreversible after 96 erg/mm². The rate of synthesis of DNA in the presence of chloramphenicol was shown to be very close to the rate shown by bacteria incubated in the absence of chloramphenicol, indicating that all active sites of replication remaining after UV-damage remain active in the presence of chloramphenicol, as expected if the limiting effect of chloramphenicol is on initiation at the chromosomal origin and not due to reduction in rate of DNA replication.A much lower concentration of chloramphenicol (2 μg/ml) blocking only the chloramphenicol-sensitive event in control of DNA replication described by Ward and Glaser¹⁵, imposed a limitation in DNA accumulation in the culture of somewhat less than a doubling, as would be expected if the antibiotic at this concentration does not block the chloramphenicol-resistant control event. DNA degradation occured with incubation of bacteria given a UV-dose sufficient to inactivate all active DNA replication sites on their chromosomes, when in medium containing chloramphenicol concentrations (above 20 μg/ml) sufficient to block the chloramphenicol-resistant control event. Such breakdown resulted in death. The damage responsible for such death and DNA breakdown was not photoreversible after this dose, supporting the hypothesis that breakdown results from non-photoreversible inactivation of active DNA replication sites. This was in contrast to increased death in UV-damaged bacteria promoted by nalidixic acid, a specific inhibitor of DNA replication, which could be prevented in part by light exposure after the same UV-dose.     

Effect of 6-(<Emphasis Type="Italic">p</Emphasis>-Hydroxyphenyl)-azouracil on <Emphasis Type="Italic">B. subtilis</Emphasis> DNA Polymerases

DNA replication in Bacillus subtilis^1,2 and other Gram-positive organisms³ is specifically inhibited by 6-(p-hydroxyphenyl)-azouracil (HPUra). The site of action of this compound has not so far been identified, but important progress was made by Brown et al.⁴, who studied the effect of HPUra on DNA synthesis in B. subtilis cells made permeable to externally supplied deoxynucleoside triphosphates by treatment with toluene. In this in vitro system, HPUra had no inhibitory effect when added alone, but in the presence of NADPH or dithiothreitol (DTT) the drug was reduced to a colourless form which specifically inhibited DNA synthesis.     

A novel Escherichia coli mutant capable of DNA replication in the absence of protein synthesis.

An Escherichia coli mutant capable of continued DNA synthesis in the presence of chloramphenicol has been isolated by an autoradiographic technique. The DNA synthesis represents semiconservative replication of E. coli DNA. It can occur in the presence of chloramphenicol or in the absence of essential amino acids, but not in the presence of an RNA synthesis inhibitor, rifampin. The mutant, termed constitutive stable DNA replication (Sdr^c) mutant, appears to grow normally at 37 °C with a slightly slower growth rate than that of the parental strain. DNA replication in the mutant occurs at a reduced rate after 60 minutes in the absence of protein synthesis and continues linearly for several hours thereafter. This distinct slowdown in the DNA replication rate is due to a reduced rate of DNA synthesis in all the cells in the population. Constitutive stable DNA replication appears to require the dnaA and dnaC gene products. The sdr^c mutation has been mapped near the pro-lac region of the E. coli chromosome. The mutation is recessive. Autoradiographic experiments have ruled out the possibility of multiple initiations during a cell cycle. The implication of the above findings is discussed in terms of the regulation of chromosome replication in E. coli.     

On the termination of the DNA replication cycle in Escherichia coli.

The initiation of the DNA replication cycle in Escherichia coli requires protein synthesis. Marunouchi &; Messer (1973) have hypothesized that an additional protein synthesis step is required for the replication of the terminal segment of the chromosome, and that replication of this segment is a prerequisite for subsequent cell division. We have not confirmed the existence of a unique terminal segment using a protocol designed to label the hypothesized segment with [³H]dThd². Our protocol avoids the increased incorporation of [³H]dThd into DNA caused by abrupt increases in temperature, a complication implicit in the technique of Marunouchi &; Messer (1973).Treatment with nalidixic acid (an inhibitor of semiconservative DNA synthesis) in sufficient concentration to prevent replication of the postulated terminal segment prevents cell division but also causes loss of viability. This makes it difficult to correlate the effect of nalidixic acid on cell division with DNA synthesis inhibition alone.     

Direct Inhibition of <Emphasis Type="Italic">Col</Emphasis> E<Subscript>1</Subscript> Plasmid DNA Replication in <Emphasis Type="Italic">Escherichia coli</Emphasis> by Rifampicin

COLICINOGENIC factor E₁ (Col E₁) is a small bacterial plasmid (4.2×10⁶ daltons) present in colicinogenic strains of Escherichia coli¹ to the extent of about twenty-four copies per cell (Clewell and Helinski, unpublished results), which continues to replicate in the presence of high levels of chloramphenicol, a specific inhibitor of protein synthesis, although the chromosome only completes current rounds of replication and ceases (Clewell and Helinski, unpublished results). The average rate of Col E₁ semiconservative replication in the absence of protein synthesis is, in certain conditions, faster than (as much as eight times) the normal rate of synthesis (Clewell, unpublished results). Replication continues for 10–15 h after the addition of chloramphenicol, resulting in nearly 3,000 copies of Col E¹ DNA per cell. We are taking advantage of this system to study the effects of a number of antibiotics on DNA replication and now report evidence that rifampicin (an active semisynthetic derivative of rifamycin B)², an antibiotic known specifically to inhibit bacterial DNA dependent RNA polymerase^3–6, has a dramatic inhibitory effect on Col E¹ DNA replication.     

Efficient long DNA gap-filling in a mammalian cell-free system: A potential new in vitro DNA replication assay

In vitro assay of mammalian DNA replication has been variously approached. Using gapped circular duplex substrates containing a 500-base single-stranded DNA region, we have constructed a mammalian cell-free system in which physiological DNA replication may be reproduced. Reaction of the gapped plasmid substrate with crude extracts of human HeLaS3 cells induces efficient DNA synthesis in vitro. The induced synthesis was strongly inhibited by aphidicolin and completely depended on dNTP added to the system. In cell extracts in which PCNA was depleted step-wise by immunoprecipitation, DNA synthesis was accordingly reduced. These data suggest that replicative DNA polymerases, particularly pol delta, may chiefly function in this system. Furthermore, DNA synthesis is made quantifiable in this system, which enables us to evaluate the efficiency of DNA replication induced. Our system sensitively and quantitatively detected the reduction of the DNA replication efficiency in the DNA substrates damaged by oxidation or UV cross-linking and in the presence of a potent chain terminator, ara-CTP. The quantitative assessment of mammalian DNA replication may provide various advantages not only in basic research but also in drug development.     

Bacteriophage phi X174 RF DNA replication in vivo. A study by electron microscopy.

We have investigated bacteriophage φX174 RF ² DNA replication by electron microscopy. Three different, types of replicative intermediates were observed: rolling circles, partially duplex DNA circles and structures consisting of two DNA circles connected at a single point.Rolling circles with a single-stranded or partially double-stranded DNA tail were both observed. After cleavage of the rolling circles with the restriction endonuclease from Providentia stuartii 164 (PstI) the startpoint of rolling circle replication could be located at 21 map units from the PstI cleavage site in agreement with the previously determined position of the origin of φX RF DNA replication.Partially duplex DNA circles consist of circular viral DNA strands and incomplete complementary DNA strands. After cleavage of these molecules with PstI information about the startpoints of the synthesis of the complementary DNA strand was obtained.The connected DNA circles always contain one completely double-stranded DNA circle whereas the other circle consists of either single-stranded, partially duplex or completely duplex DNA.Part of the duplex-to-duplex DNA circles represent the well-known figure eight or catenated circular dimers. The other connected DNA circles presumably represent replication intermediates which arise by the association of the end of the genome length tail of the rolling circle with the origin-terminus region. This is suggested by the fact that the point of contact between the two DNA circles is located at approximately 21 map units from the Pst1 cleavage site, i.e. at the origin-terminus region of the φX genome. The connected DNA circles may be intermediates in the circularization and cleavage of the genome-length tail of the rolling circles in vivo.A model for φX174 RF DNA replication in vivo summarizing the data obtained by biochemical (Baas et al., 1978) and electron microscopic analysis of replicative intermediates is presented (Fig. 9).     

The role of HERC2 and RNF8 ubiquitin E3 ligases in the promotion of translesion DNA synthesis in the chicken DT40 cell line

The replicative DNA polymerases are generally blocked by template DNA damage. The resulting replication arrest can be released by one of two post-replication repair (PRR) pathways, translesion DNA synthesis (TLS) and template switching by homologous recombination (HR). The HERC2 ubiquitin ligase plays a role in homologous recombination by facilitating the assembly of the Ubc13 ubiquitin-conjugating enzyme with the RNF8 ubiquitin ligase. To explore the role of HERC2 and RNF8 in PRR, we examined immunoglobulin diversification in chicken DT40 cells deficient in HERC2 and RNF8. Unexpectedly, the HERC2^−/− and RNF8^−/− cells and HERC2^−/−/RNF8^−/− double mutant cells exhibit a significant reduction in the rate of immunoglobulin (Ig) hypermutation, compared to wild-type cells. Further, the HERC2^−/− and RNF8^−/− mutants exhibit defective maintenance of replication fork progression immediately after exposure to UV while retaining proficient post-replicative gap filling. These mutants are both proficient in mono-ubiquitination of PCNA. Taken together, these results suggest that HERC2 and RNF8 promote TLS past abasic sites and UV-lesions at or very close to stalled replication forks.     

Pre-Meiotic DNA Synthesis and Recombination in CHLAMYDOMONAS REINHARDI

The time of the pre-meiotic S-period was determined by ³²P incorporation in synchronously germinating zygospores of Chlamydomonas reinhardi at six and one-half to seven hours after the beginning of germination. Phenethyl alcohol treatment caused death of zygospores at a period one hour before the S-period, and also during meiotic prophase. Recombination between arg-1 and arg-2 was increased by treatment with phenethyl alcohol or mitomycin C at a time between the first sensitive period to phenethyl alcohol and the S-period. Actinomycin D caused an increase in recombination at the time of this sensitive period. FUdR, nalidixic acid and hydroxurea all cause a decrease in recombination when applied during S-period, and have no effect earlier. These results are explained by postulating (1) that the units of delayed premeiotic replication are whole replicons, and (2) that the amount of recombination is proportional to the number of replicons in which synthesis is delayed. It is suggested that the control of DNA replication controls the distribution of recombination events.     

The effect of sodium butyrate on Tipula iridescent virus synthesis in insect suspension cell cultures

The effect of sodium butyrate on Tipula iridescent virus (TIV) synthesis in suspension-cultured cells of Estigmene acrea was investigated. Sodium butyrate reduces viral-induced cell fusion but this is reversible with the removal of butyrate. At 7 mM sodium butyrate, TIV replicates in cells within 8 hr, but does not replicate in this time with 10–20 mm butyrate in the cell medium; cells so treated contain large vesicles with inoculum. Upon removal of the inhibitor, TIV replication appears normal, but large inoculum vesicles can still be found in the cytoplasm, and many infected cells have highly condensed chromatin in their nuclei. Sodium butyrate causes a lag of at least 2 hr in viral DNA synthesis as detected by [³H]thymidine incorporation into viroplasmic centres and at 7 mm butyrate viral DNA synthesis is reduced by 50–60%. In comparison, butyrate at 7 and 10 mm concentration does not inhibit host DNA synthesis, but at 15 and 20 mm, nuclear DNA synthesis is markedly reduced.     

Defective DNA Synthesis in Permeabilized Yeast Mutants

THE simple eukaryote, Saccharomyces cerevisiae, is suitable for combined genetic and biochemical analysis of the cell division cycle. More than forty temperature-sensitive mutants of S. cerevisiae defective in fifteen genes that control various steps of the yeast cell cycle have been detected by screening a collection of mutants with time-lapse photomicroscopy¹. Mutations in two genes, cdc4 and cdc8, result in defective DNA synthesis at the restrictive temperature². The product of cdc8 is apparently required throughout the period of DNA synthesis, because if a strain defective in this gene is shifted to 36° C within the S period, DNA replication ceases. In contrast, the product of cdc4 is apparently required only at the initiation of DNA synthesis because when a strain carrying a defect in this gene is shifted to 36° C, DNA replication already in progress is not impaired. Cells defective in cdc4, however, fail to initiate new rounds of DNA synthesis at the restrictive temperature. Based on these observations the DNA mutants have been tentatively classified as defective in DNA replication (cdc8) and in the initiation of DNA synthesis (cdc4).     

Integration of Deoxyribonuclease-Treated DNA in Bacillus subtilis Transformation

Normal preparations of B. subtilis DNA have weight average native molecular weights of 10 to 30 x 10⁶. For any given preparation the upper and lower 95% size limits may differ by a factor of ten or more. Single-stranded molecular weights indicate an average of 1 to 4 breaks per single strand of the native DNA. The reduction in transforming activity and viscosity following DNAase I digestion can be accounted for by a direct relationship between the transforming activity of a DNA and its single-stranded molecular weight. Uptake studies with DNAase I treated heavy (²H¹⁵N ³H) DNA show that single strand breaks inhibit integration less than transformation. A provisional estimate of the size of the integrated region based on correlating the single strand size of the donor-recipient complex with the donor-recipient density differences following alkali denaturation came to 1530 nucleotides. Using a competent, nonleaky thymine-requiring strain of B. subtilis grown in 5-BU medium before and after transformation, it was shown that (a) No detectable amount of DNA synthesis is necessary for the initial stages of integration, (b) Cells which have recently been replicating DNA are not competent. (c) Cells containing donor DNA show a lag in DNA replication following transformation, (d) When donor DNA is replicated it initially appears in a density region between light and hybrid. This indicates that it includes the transition point formed at the time of reinitiation of DNA synthesis in the presence of 5-BU following transformation. A model is proposed in which donor DNA is integrated at the stationary growing point of the competent cell, which is in a state of suspended DNA synthesis.     

Cell cycle defects in polyhomeotic mutants are caused by abrogation of the DNA damage checkpoint

Polycomb group (PcG) genes are required for heritable silencing of target genes. Many PcG mutants have chromatin bridges and other mitotic defects in early embryos. These phenotypes can arise from defects in S phase or mitosis, so the phenotype does not show when PcG proteins act in cell cycle regulation. We analyzed the cell cycle role of the proximal subunit of Polyhomeotic (PhP) in Drosophila. Time-lapse imaging reveals that chromatin bridges formed during mitosis are able to resolve but sometimes result in chromosome breakage. Chromosome bridging is also observed in canonical cell cycles occurring in larval brains and is therefore not unique to the rapid embryonic cycles. PhP colocalizes with chromatin in S phase but not in mitosis in early embryos, indicating a direct role in DNA synthesis. Time lapse imaging of ph^p mutants reveals an acceleration of S phase, showing that ph^p regulates S phase length. Like ph^p mutations, mutations in DNA damage checkpoints result in S phase acceleration. Consistent with this model, mutations in ph do not affect DNA synthesis rates, but exhibit impaired ability to block cell cycle progression following exposure to gamma-rays. Our data show that the mitotic defects of ph^p are caused by defects in the DNA damage response that occurs after DNA replication in S phase, and we propose that PhP has a direct role in DNA damage repair.     

A genetic study based on PCNA-ubiquitin fusions reveals no requirement for PCNA polyubiquitylation in DNA damage tolerance

Post-translational modifications of Proliferating Cell Nuclear Antigen (PCNA) play a key role in regulating the bypass of DNA lesions during DNA replication. PCNA can be monoubiquitylated at lysine 164 by the RAD6-RAD18 ubiquitin ligase complex. Through this modification, PCNA can interact with low fidelity Y family DNA polymerases to promote translesion synthesis. Monoubiquitylated PCNA can be polyubiquitylated on lysine 63 of ubiquitin by a further ubiquitin-conjugating complex. This modification promotes a template switching bypass process in yeast, while its role in higher eukaryotes is less clear.We investigated the function of PCNA ubiquitylation using a PCNA^K164R mutant DT40 chicken B lymphoblastoma cell line, which is hypersensitive to DNA damaging agents such as methyl methanesulfonate (MMS), cisplatin or ultraviolet radiation (UV) due to the loss of PCNA modifications. In the PCNA^K164R mutant we also detected cell cycle arrest following UV treatment, a reduced rate of damage bypass through translesion DNA synthesis on synthetic UV photoproducts, and an increased rate of genomic mutagenesis following MMS treatment. PCNA-ubiquitin fusion proteins have been reported to mimic endogenous PCNA ubiquitylation. We found that the stable expression of a PCNA^K164R-ubiquitin fusion protein fully or partially rescued the observed defects of the PCNA^K164R mutant. The expression of a PCNA^K164R-ubiquitin^K63R fusion protein, on which the formation of lysine 63-linked polyubiquitin chains is not possible, similarly rescued the cell cycle arrest, DNA damage sensitivity, reduction of translesion synthesis and increase of MMS-induced genomic mutagenesis. Template switching bypass was not affected by the genetic elimination of PCNA polyubiquitylation, but it was reduced in the absence of the recombination proteins BRCA1 or XRCC3. Our study found no requirement for PCNA polyubiquitylation to protect cells from replication-stalling DNA damage.