T4-endonuclease V-sensitive sites in DNA from ultraviolet-irradiated human cells.

Human diploid cells (WI38) were pre-labeled with 32Pi, exposed to ultraviolet irradiation and then pulse labeled with [3H]thymidine. The extracted DNA from these cells was subsequently treated with the T4-endonuclease V, an enzyme which specifically nicks DNA strands at positions adjacent to pyrimidine dimers. Sedimentation in alkaline sucrose gradients revealed that the DNA synthesized after irradiation, as well as that made before, contained endonuclease-sensitive sites. Our results suggest that pyrimidine dimers are transferred from parental to daughter DNA strands during post-irradiation incubation. Sedimentation in neutral sucrose gradients showed that the molecular weight of native DNA was not affected by the endonuclease treatment, suggesting that the gaps appearing in daughter strands after irradiation are not opposite dimers or that the enzyme cannot recognize dimers in the gap regions.     

Replication of the lagging strand: a concert of at least 23 polypeptides.

DNA replication is one of the most important events in living cells, and it is still a key problem how the DNA replication machinery works in its details. A replication fork has to be a very dynamic apparatus since frequent DNA polymerase switches from the initiating DNA polymerase alpha to the processive elongating DNA polymerase delta occur at the leading strand (about 8 x 10(4) fold on both strands in one replication round) as well as at the lagging strand (about 2 x 10(7) fold on both strands in one replication round) in mammalian cells. Lagging strand replication involves a very complex set of interacting proteins that are able to frequently initiate, elongate and process Okazaki fragments of 180 bp. Moreover, key proteins of this important process appear to be controlled by S-phase check-point proteins. It became furthermore clear in the last few years that DNA replication cannot be considered uncoupled from DNA repair, another very important event for any living organism. The reconstitution of nucleotide excision repair and base excision repair in vitro with purified components clearly showed that the DNA synthesis machinery of both of these macromolecular events are similar and do share many components of the lagging strand DNA synthesis machinery. In this minireview we summarize our current knowledge of the components involved in the execution and regulation of DNA replication at the lagging strand of the replication fork.     

Two pathways of division inhibition in UV-irradiated E. coli

We have investigated the mechanism of division inhibition in E. coli following UV-irradiation or nalidixic acid treatment. After UV, two separate mechanisms, both dependent upon recA+, appear to block division. One mechanism is dependent upon sfiA and sfiB, is inhibited by low levels (4 micrograms/ml) of rifamycin and is expressed in tif mutants at 42 degrees C. The second mechanism is independent of sfiA, and sfiB, is resistant to rifamycin and does not occur in cells lacking DNA replication forks. We suggest that this second mechanism is the result of the failure to terminate DNA replication in inhibited cells. Nalidixic acid inhibition of cell division also appears to involve both mechanisms but as found previously replication forks are also necessary to induce the sfi pathway.     

The timing of UV mutagenesis in yeast: continuing mutation in an excision-defective (rad1-1) strain.

Summary Mutation induction by ultraviolet light was studied in excision-defective (rad1-1) strains of Saccharomyces cerevisiae. Information about the timing of mutations in relation to postirradiation DNA replication was obtained. The experimental system involved pedigree analysis of G1 diploid cells and subsequent tetrad analysis of the mitotic segregants to detect mutations.The mutation pattern of rad1-1 strains differed from that of wild type in two respects: (1) few or none of the mutations affected both strands of the DNA, (2) mutations appeared as frequently in the second postirradiation mitotic generation as in the first.The data have led to the following conclusions about the mutation process in excision-defective (rad1-1) yeast: (a) Mutations are not fixed prior to the first postirradiation round of DNA replication. (b) Unexcised thymine dimers persist as mutagenic lesions through repeated postirradiation cell divisions. (c) Heteroduplex repair is involved in the mutation process. (d) Overlapping daughter-strand gaps are not a prerequisite for mutation. (e) The results provide no evidence that error-prone repair in this strain is inducible rather than constitutive. The data also show that (f) all 2-strand mutations (whole-colony mutants) induced in yeast by exposure to low levels of ultraviolet light are associated with excision repair, and that (g) essentially all lesions induced in excision-proficient strains have been excised at the time of the second round of postirradiation DNA synthesis.On leave of absence from the Department of Genetics, University of Edinburgh, West Mains Road, Edinburgh, Scotland EH9 3JN     

Pachytene Arrest and Other Meiotic Effects of the Start Mutations in Saccharomyes Cerevisiae

Mutations in the Start class of cell division cycle genes (CDC28, CDC36 and CDC39) define the point in the G1 phase of the vegetative cycle at which the cell becomes committed to completing another round of cell division. Genetic, cytological and biochemical data demonstrate that these mutations cause meiotic cells to become arrested at pachytene following completion of both chromosomal DNA replication and spindle pole body (SPB) duplication. In contrast these mutations have previously been found to cause arrest of the mitotic cell cycle prior to either of these landmark events, so the role of the Start genes in these events during vegetative growth must be indirect. Our observations are consistent with the hypothesis that CDC28, CDC36 and CDC39 are required for irreversible commitment to nuclear division in both the mitotic and meiotic pathways. CDC28 was additionally found to be required for the SPB separation that precedes spindle formation in preparation for the second meiotic division. Cytological and genetic analyses of this requirement revealed both that such separation may fail independently at either SPB and that ascospore formation can proceed independently of SPB separation.     

Misincorporation rate and type on the leading and lagging strands of UV-damaged DNA.

We have examined the fidelity of replication of the leading and lagging strands of UV-irradiated DNA by using an EBV-derived shuttle vector system which contains as marker gene for mutation analysis the bacterial gpt gene in both orientations relative to the EBV oriP. Human cells stably transformed with this vector were UV irradiated and gpt mutation rate and type were analysed. An increased mutagenicity associated with UV irradiation was observed, but the average error frequency was unaffected by the direction of replication of the target gene. Some variability by position and sequence context of leading and lagging strand errors was detected, suggesting that the different architecture of the replication complex for the two strands might, to some extent, affect mutation spectra. The comparable fidelity of translesion replication on the leading and lagging strands is in agreement with the current model for eukaryotic replication that postulates the simultaneous synthesis of both strands by a DNA polymerase with a proof-reading exonuclease.     

Effects of ultraviolet irradiation on the rate and sequence of DNA replication in synchronized Chinese hamster cells.

The effects of ultraviolet light (UV) irradiation on the rate of DNA replication in synchronized Chinese hamster ovary (CHO) cells were investigated. A technique for measuring semiconservative DNA replication was employed that involved growing the cells in medium containing 5-bromodeoxyuridine and subsequently determining the amount of DNA that acquired hybrid buoyant density in CsCl density gradients. One of the advantages of this technique was that it allowed a characterization of the extent of DNA replication as well as rate after irradiation. It was found that while there was a dose-dependent reduction in the rate of DNA replication following UV-irradiation, doses of up to 10 J/m2 (which produce many dimers per replication) did not prevent the ultimate replication of the entire genome. Hence, we conclude that dimers cannot be absolute blocks to DNA replication. In order to account for the total genome replication observed, a mechanism must exist that allows genome replication between dimers. The degree of reduction in the rate of replication by UV was the same whether the cells were irradiated at the G1-S boundary or 1 h into S-phase. Previous work had shown that cells in early S-phase are considerably more sensitive to UV than cells at the G1-S boundary. Experiments specifically designed to test for reiterative replication showed that UV does not induce a second round of DNA replication within the same S-phase.     

Structure of F-actin needles from extracts of sea urchin oocytes

The mouse L-cell line LD maintains its mitochondrial DNA genome in the form of a head-to-tail unicircular dimer of the monomeric 16,000 base-pair species. This situation permits a comparison of the mechanism of replication of this dimeric molecule with our previous studies of replication of monomeric mouse L-cell mitochondrial DNA. Whereas monomeric mitochondrial DNA requires about one hour for a round of replication, the dimeric molecule requires almost three hours. Denaturing agarose gel electrophoretic analyses of replicative intermediates reveals several discrete size classes of partially replicated daughter strands of dimeric mitochondrial DNA. This suggests that replication occurs with specific discontinuities in the rate of daughter strand synthesis. The strand specificity of these daughter strands was determined by hybridization with ³²P-labeled DNA representing either the heavy or light strand mitochondrial DNA sequence. The sizes and strand specificities of these discrete daughter strands indicate that the same set of control sequences is functional in both dimer and monomer mitochondrial DNA replication.Immediately following a round of replication, the majority of dimeric mitochondrial DNA molecules contain displacement loops, as assessed by their sensitivity to nicking within the displaced DNA strand by single-strand DNA specific S₁ nuclease under conditions which leave supercoiled DNA intact. This result is in contrast with the conformation of newly replicated monomeric mitochondrial DNA molecules, which lack both superhelical turns and displacement loops. This indicates that dimeric mitochondrial DNA proceeds through a different series of post-replicative processing steps than does monomeric mitochondrial DNA. We postulate that intermediates at late stages of dimeric mitochondrial DNA replication contain displacement loops which remain intact following closure of the full-length daughter strands.     

Regulation of Deoxyribonucleic Acid Replication and Cell Division in Escherichia coli B/r

Synchronous cultures of Escherichia coli strain B/r were used to investigate the relationship between deoxyribonucleic acid (DNA) replication and cell division. We have determined that terminal steps in division can proceed in the absence of DNA synthesis. Inhibition of DNA replication with nalidixic acid prior to the start of a new round of replication does not stop cell division, which indicates that the start of the round is not essential in triggering cell division. Inhibition of DNA replication at any time prior to the termination of a round of replication completely blocks cell division, which suggests that there may be a link between the end of the replication cycle and the commitment of the cell to divide. Studies that use a temperature-sensitive mutant which is unable to synthesize DNA at the nonpermissive temperature are in complete agreement with those that use nalidixic acid to inhibit DNA synthesis. This adds support to the idea that the treatments employed limit their action to DNA synthesis. Investigation of minicell production indicates that the production of minicells is blocked when DNA synthesis is inhibited with nalidixic acid. Although nuclear segregation is not required for cell division, DNA synthesis is still required to trigger division. The evidence presented suggests strongly that (i) DNA synthesis is essential for cell division, (ii) the end of a round of replication triggers cell division, and (iii) there is considerable time lapse (one-half generation) between the completion of a round of DNA replication and physical separation of the cells.     

Immortal DNA or epigenetic signature ?

During mitosis each daughter cell inherits a full copy of the maternal genomic material. DNA replication, however, is an imprecise process, thus errors can arise resulting in potentially deleterious mutations over extended rounds of cell division and these may lead to cancinogenesis. Over thirty years ago, J. Cairns proposed that a cell could avoid the accumulation of mutations arising from DNA replication if all template DNA strands are inherited in one daughter cell during cell division, thus giving rise to the notion of < immortal > DNA strands. In this model the stem cells would retain the template DNA (older) strands. Proving or disproving this notion experimentally has been challenging. Further, it has recently become apparent that epigenetic regulation of gene expression plays a critical role in governing cell states, self-renewal and differentiation. In light of these data, can the phenomenon on template DNA strand segregation also reflect this epigenetic signature? In this review we explore these notions, discuss the evidence in support of this theory, the implications, and some of the mechanisms which could explain this phenomenon.     

Inhibition of initiation of simian virus 40 DNA replication during acute response of cells irradiated by ultraviolet light.

To study the mechanism by which ultraviolet (UV) light inhibits DNA replication, we examined the effects of UV 254 nm irradiation on the replication of simian virus 40 (SV40) DNA and SV40-based plasmid in monkey cells. The study was designed to determine the relative contributions made by inhibition of replication initiation and chain elongation to the immediate inhibition of DNA replication following UV irradiation. We used two-dimensional neutral-alkaline electrophoresis to examine the behaviour of replication intermediates unambiguously. Kinetic analysis using this technique showed that initiation of replication started to decline at 15 min post-irradiation. When the pulse label incorporated in SV40 replication intermediates before irradiation was chased for 1 h, most of the label was found in mature Form I and II molecules. This indicated that replication elongation took place on damaged template. We also used a transfection technique to show that heavily irradiated plasmids replicated efficiently in unirradiated transfected cells. By the transfection technique, we observed that UV irradiation of host cells dose-dependently inhibited replication of transfected non-irradiated plasmids, suggesting that the inhibition of DNA replication is due to a global change in cellular physiology induced by UV. This change was also apparent from poor staining of the chromatin by fluorescent-DNA-binding dyes immediately after UV irradiation of intact cells. We conclude that a significant fraction of chain elongation proceeds on damaged templates and DNA replication during the acute response of cells irradiated with UV is mainly controlled by the inhibition of replication initiation.     

Chromosome methylation and measurement of faithful, once and only once per cell cycle chromosome replication in Caulobacter crescentus

Caulobacter crescentus exhibits cell-type-specific control of chromosome replication and DNA methylation. Asymmetric cell division yields a replicating stalked cell and a nonreplicating swarmer cell. The motile swarmer cell must differentiate into a sessile stalked cell in order to replicate and execute asymmetric cell division. This program of cell division implies that chromosome replication initiates in the stalked cell only once per cell cycle. DNA methylation is restricted to the predivisional cell stage, and since DNA synthesis produces an unmethylated nascent strand, late DNA methylation also implies that DNA near the replication origin remains hemimethylated longer than DNA located further away. In this report, both assumptions are tested with an engineered Tn5-based transposon, Tn5Omega-MP. This allows a sensitive Southern blot assay that measures fully methylated, hemimethylated, and unmethylated DNA duplexes. Tn5Omega-MP was placed at 11 sites around the chromosome and it was clearly demonstrated that Tn5Omega-MP DNA near the replication origin remained hemimethylated longer than DNA located further away. One Tn5Omega-MP placed near the replication origin revealed small but detectable amounts of unmethylated duplex DNA in replicating stalked cells. Extra DNA synthesis produces a second unmethylated nascent strand. Therefore, measurement of unmethylated DNA is a critical test of the "once and only once per cell cycle" rule of chromosome replication in C. crescentus. Fewer than 1 in 1,000 stalked cells prematurely initiate a second round of chromosome replication. The implications for very precise negative control of chromosome replication are discussed with respect to the bacterial cell cycle.     

Inhibition of repair of UV-damaged DNA by caffeine and mutation induction in Chinese hamster cells

The effect of caffeine on UV-irradiated Chinese hamster cells in vitro was studied on the cellular and molecular levels. Caffeine (1 mM) was shown to decrease the colony-forming ability and the frequencies of spontaneous and UV-induced mutations in Chinese hamster cells. The effect of caffeine in reducing the frequency of UV-induced mutations was demonstrated only if caffeine was present in the culture medium during the first post-irradiation cell division. Using alkaline sucrose gradient centrifugation, both parental and newly synthesized DNA in UV-irradiated and unirradiated cells were studied in the presence and absence of caffeine. Caffeine affected the sedimentation profile of DNA synthesized in UV-irradiated cells but not in unirradiated cells. Caffeine had no apparent effect on the incorporation of [³H]-thymidine into DNA of control or UV-irradiated cells, nor on the small amount of excision of UV-induced pyrimidine dimers. These results may be interpreted by a hypothesis that caffeine inhibits a certain S-phase specific, post-replication, dark-repair mechanism. The hamster and perhaps other rodent cells exposed to low doses of UV are capable of DNA replication, by-passing the non-excised pyrimidine dimers. This postulated repair process probably involves de novo DNA synthesis to seal the gaps in the nascent strand. This repair may be also responsible for the enzymatic production of mutations.     

Yeast pip3/mec3 mutants fail to delay entry into S phase and to slow DNA replication in response to DNA damage, and they define a functional link between Mec3 and DNA primase.

The catalytic DNA primase subunit of the DNA polymerase alpha-primase complex is encoded by the essential PRI1 gene in Saccharomyces cerevisiae. To identify factors that functionally interact with yeast DNA primase in living cells, we developed a genetic screen for mutants that are lethal at the permissive temperature in a cold-sensitive pril-2 genetic background. Twenty-four recessive mutations belonging to seven complementation groups were identified. Some mutants showed additional phenotypes, such as increased sensitivity to UV irradiation, methyl methanesulfonate, and hydroxyurea, that were suggestive of defects in DNA repair and/or checkpoint mechanisms. We have cloned and characterized the gene of one complementation group, PIP3, whose product is necessary both for delaying entry into S phase or mitosis when cells are UV irradiated in G1 or G2 phase and for lowering the rate of ongoing DNA synthesis in the presence of methyl methanesulfonate. PIP3 turned out to be the MEC3 gene, previously identified as a component of the G2 DNA damage checkpoint. The finding that Mec3 is also required for the G1- and S-phase DNA damage checkpoints, together with the analysis of genetic interactions between a mec3 null allele and several conditional DNA replication mutations at the permissive temperature, suggests that Mec3 could be part of a mechanism coupling DNA replication with repair of DNA damage, and DNA primase might be involved in this process.     

Fidelity of replication of the leading and the lagging DNA strands opposite N-methyl-N-nitrosourea-induced DNA damage in human cells.

Semi-conservative replication of double-stranded DNA in eukaryotic cells is an asymmetric process involving leading and lagging strand synthesis and different DNA polymerases. We report a study to analyze the effect of these asymmetries when the replication machinery encounters alkylation-induced DNA adducts. The model system is an EBV-derived shuttle vector which replicates in synchrony with the host human cells and carries as marker gene the bacterial gpt gene. A preferential distribution of N-methyl-N-nitrosourea (MNU)-induced mutations in the non transcribed DNA strand of the shuttle vector pF1-EBV was previously reported. The hypermutated strand was the leading strand. To test whether the different fidelity of DNA polymerases synthesizing the leading and the lagging strands might contribute to MNU-induced mutation distribution the mutagenesis study was repeated on the shuttle vector pTF-EBV which contains the gpt gene in the inverted orientation. We show that the base substitution error rates on an alkylated substrate are similar for the replication of the leading and lagging strands. Moreover, we present evidence that the fidelity of replication opposite O6-methylguanine adducts of both the leading and lagging strands is not affected by the 3' flanking base. The preferential targeting of mutations after replication of alkylated DNA is mainly driven by the base at the 5' side of the G residues.     

The stage of chromosome duplication in the cell cycle as revealed by X-ray breakage and 3H-thymidine labeling

By means of combined experiments of X-irradiation and ³H-thymidine labeling of the chromosomes which are in the phase of synthesis, and the subsequent analysis at metaphase on the autoradiographs of the chromosomal damage induced during interphase, it was shown that in somatic cells from a quasi-diploid Chinese hamster line cultured in vitro the chromosomes change their response to radiation from single (chromosome type aberrations) to double (chromatid type aberrations) in late G₁. These results are interpreted to indicate that the chromosome splits into two chromatids in G₁, before DNA replication. — By extending the observations at the second metaphase after irradiation, it was also seen that cells irradiated while in G₂ or late S when they reach the second post-irradiation mitosis still exhibit, beside chromosome type aberrations, many chromatid exchanges, some of which are labeled. Two hypotheses are suggested to account for this unexpected reappearance of chromatid aberrations at the second post-irradiation division. The first hypothesis is that they arise from half-chromatid aberrations. The second hypothesis, which derives from a new interpretation of the mechanisms of production of chromosome aberrations recently forwarded by Evans, is that they arise from gaps or achromatic lesions which undergo, as the cells go through the next cycle, a two-step repair process culminating in the production of aberrations.This work was supported in part by grant No. RH-00304 from the Division of Radiological Health, Bureau of State Services, Public Health Service, U.S.A.     

Random-choice replication of extrachromosomal bovine papillomavirus (BPV) molecules in heterogeneous, clonally derived BPV-infected cell lines.

Using fluorescence in situ hybridization and Southern blot analysis, we show that three clonally derived cell lines transformed with bovine papillomavirus (BPV), including ID13, the cell line commonly employed for BPV replication studies, are heterogeneous populations having extensive cell-to-cell variation in both the distribution and amount of BPV DNA. Different subclones of ID13 were found to differ in the form and amount of BPV DNA they contain. Most subclones showed no detectable BPV sequences; some contained either extrachromosomal BPV molecules distributed throughout the nucleus or BPV sequences integrated at discrete chromosomal sites, while others contained both integrated and plasmid forms. The results of density gradient analysis of BPV DNA from individual homogeneous subclones showed replication of the extrachromosomal BPV plasmids in a random-choice mode. In all cell lines studied, the presence after one round of chromosomal DNA replication of unreplicated BPV DNA and of BPV DNA having two postreplicative strands was independent of the presence of high-BPV-copy-number ("jackpot") cells. Our results substantiate the earlier conclusion that extrachromosomal BPV molecules replicate randomly and not according to a once-per-cell-cycle mechanism.     

Caulobacter crescentus fatty acid-dependent cell cycle mutant.

A fatty acid auxotroph of Caulobacter crescentus, AE6001, which displays a strict requirement for unsaturated fatty acids to grow on glucose as the carbon source has been isolated. Starvation of AE6001 for unsaturated fatty acids resulted in a block in the cell cycle. Starved cultures accumulated at the predivisional cell stage after a round of DNA replication had been completed and after a flagellum had been assembled at the pole of the cell. Cell division and cell growth failed to occur probably because the mutant was unable to synthesize a membrane. An analysis of double mutants containing the fatB503 allele and other mutations in membrane biogenesis demonstrated that the cell cycle of AE6001 blocked at a homeostatic state. The addition of oleic acid to starved cultures permitted cell division and the initiation of a new round of DNA replication. The coincident block in both the initiation of DNA replication and membrane assembly, exhibited by starved cultures of this mutant, suggests that the fatB503 gene product may be involved in the coordination of these events.     

B-type cyclins CLB5 and CLB6 control the initiation of recombination and synaptonemal complex formation in yeast meiosis

BACKGROUND: The life cycle of most eukaryotic organisms includes a meiotic phase, in which diploid parental cells produce haploid gametes. During meiosis a single round of DNA replication is followed by two rounds of chromosome segregation. In the first, or reductional, division (meiosis I), which is unique to meiotic cells, homologous chromosomes segregate from one another, whereas in the second, or equational, division (Meiosis II) sister centromeres disjoin. Meiotic DNA replication precedes the initiation of recombination by programmed Spo11-dependent DNA double-strand breaks. Recent reports that meiosis-specific cohesion is established during meiotic S phase and that the length of S phase is modified by recombination factors (Spo11 and Rec8) raise the possibility that replication plays a fundamental role in the recombination process. RESULTS: To address how replication influences the initiation of recombination, we have used mutations in the B-type cyclin genes CLB5 and CLB6, which specifically prevent premeiotic replication in the yeast Saccharomyces cerevisiae. We find that clb5 and clb5 clb6 but not clb6 mutants are defective in DSB induction and prior associated changes in chromatin accessibility, heteroallelic recombination, and SC formation. The severity of these phenotypes in each mutant reflects the extent of replication impairment. CONCLUSIONS: This assemblage of phenotypes reveals roles for CLB5 and CLB6 not only in DNA replication but also in other key events of meiotic prophase. Links between the function of CLB5 and CLB6 in activating meiotic DNA replication and their effects on subsequent events are discussed.