Rate of DNA replication in the DNA synthetic period of the barley chromosomes

The rate of DNA replication, as judged by H³-thymidine incorporation, at the specific time of the S-period in chromosomes of barley (Hakata No. 2) is studied by means of autoradiography.In the barley chromosomes, two different DNA units with respect to replication-time are distinguishable. The early replicating DNA is replicated at least within 1 hour ab init. of the S-period, and the late replicating DNA within 1/2 to 1 hour before the end of the S-period. The replication scarcely occurs in the middle of the S-period. These evidences suggest that the replication of chromosomal DNA in the present material does, therefore, not proceed in a continuous time sequence. Topographically, the early replicating DNA is almost confined exclusively to the distal regions of the chromosomes 1 and 5, and this situation seems applicable to other chromosomes as well, whereas the late replicating DNA is close to the centromere on its both sides. Hence, the replication of chromosomal DNA does not proceed uniformly in a longitudinal sequence along the chromosomes. The interrelationships among chromosome structure in its cytological expression, replication -pattern and -time of chromosomes, and regulating mechanisms of DNA replication are discussed.     

Replication timing of large <Emphasis Type="Italic">Sorex granarius</Emphasis> (<Emphasis Type="Italic">Soricidae</Emphasis>, <Emphasis Type="Italic">Eulipotyphla</Emphasis>) telomeres

Previously, we described the unique feature of telomeric regions in Iberian shrew Sorex granarius: its telomeres have two ranges of size, very small (3.8 kb of telomeric repeats on average) and very large discontinuous telomeres (213 kb) interrupted with 18S rDNA. In this study, we have demonstrated extraordinary replication pattern of S. granarius large telomeres that have not been shown before in other studied mammal. Using the ReD-FISH procedure, we observed prolonged, through S period, large telomere replication. Furthermore, revealed ReD-FISH asymmetric signals were probably caused by partial replication of telomeres within an hour of 5-bromodeoxyuridine treatment due to the large size and special organization. We also found that in contrast to the telomeric halo from primary fibroblasts of bovine, mink, and common shrew, telomere halo of S. granarius consists of multiple loops bundled together, some of which contain rDNA. Here, we suggested several replicons firing possibly stochastic in each large telomere. Finally, we performed the TIF assay to reveal DNA damage responses at the telomeres, and along with TIF in nuclei, we found large bodies of telomeric DNA and ?-H2AX in the cytoplasm and on the surface of fibroblasts. We discuss the possibility of additional origin activation together with recombination-dependent replication pathways, mainly homologous recombination including BIR for replication fork stagnation overcoming and further S. granarius large telomere replication.     

Effects of Toluene and Phenethyl Alcohol on the Ultrastructure of Escherichia coli

Treatment of Escherichia coli cells with 0.25% toluene caused partial dissolution of the plasma membrane concomitant with displacement of nuclear material towards the cell periphery. By contrast, treatment with 0.25% phenethyl alcohol generated intracellular membranes.     

Recombination and hydroxyurea inhibition of DNA synthesis in yeast meiosis

Summary Hydroxyurea (HU) inhibits the premeiotic DNA replication and the meiotic events that follow, namely readiness, recombination commitment, haploidisation, sporulation commitment and ascus formation. Short incubations with HU (2–4 hrs) during the premeiotic replication (i.e. starting between 3 and 6.5 hrs in sporulation medium) allow the resumption of the replication at a normal rate following the removal of the drug. The other meiotic events are similarly delayed by the approximate length of the treatment. In these experiments, intragenic recombination in ade2 reached a higher level than in the controls (x1.3–2.0 in one pair of heteroalleles and x3.0–4.0 in another pair). The recombination response to short HU treatments was not observed for a pair of heteroalleles in ade2 that normally shows a high level of meiotic recombination (750 per 10⁶ cells), nor was the response observed in a pair of heteroalleles in lys2. HU treatments have almost no effect on sporulating cells from 8 hrs onwards. At 7–7.5 hrs the meiotic cells are very sensitive to the drug and even short treatments cause cell death and massive DNA degradation.     

Induction of Excessive Deoxyribonucleic Acid Synthesis in Escherichia coli by Nalidixic Acid

Prior treatment of Escherichia coli with nalidixic acid in nutritionally complete medium altered the subsequent pattern of deoxyribonucleic acid (DNA) synthesis normally observed in nutritionally deficient medium. Transfer of E. coli 15 TAU to an amino acid- and pyrimidine-deficient medium usually resulted in a 40 to 50% increase in DNA content. Previous treatment with nalidixic acid caused a 200 to 300% increase in DNA content under these conditions. The extent of this DNA synthesis depended on the duration of prior exposure to nalidixic acid. The maximal rate of synthesis was obtained after a 40- to 60-min exposure to nalidixic acid and was two to three times that of the control. The induction of this excessive DNA synthesis was prevented by chloramphenicol or phenethyl alcohol, but the synthesis of this DNA was only partially sensitive to these agents. With E. coli TAU-bar, the rate of DNA synthesis, after removal of nalidixic acid, was similar to that of E. coli 15 TAU, but the maximal amount of DNA synthesized was 180 to 185% of that initially present. Cesium chloride density gradient analysis demonstrated that DNA synthesis after removal of nalidixic acid occurs by a semiconservative mode of replication. The density distribution of this DNA was similar to that obtained after thymine starvation. These results suggest that nalidixic acid treatment may induce additional sites for DNA synthesis in E.coli.     

The telomere resolvase of the Lyme disease spirochete,Borrelia burgdorferi,promotes DNA single-strand annealing and strand exchange

Spirochetes of the genus Borrelia include the tick-transmitted causative agents of Lyme disease and relapsing fever. They possess unusual genomes composed mainly of linear replicons terminated by closed DNA hairpin telomeres. Hairpin telomeres present an uninterrupted DNA chain to the replication machinery overcoming the ‘end-replication problem’ for the linear replicons. Hairpin telomeres are formed from inverted repeat replicated telomere junctions by the telomere resolvase, ResT. ResT uses a reaction mechanism similar to that of the type IB topoisomerases and tyrosine recombinases. We report here that ResT also possesses single-strand annealing activity and a limited ability to promote DNA strand exchange reactions on partial duplex substrates. This combination of activities suggests ResT is a nexus between the seemingly distinct processes of telomere resolution and homologous recombination. Implications for hairpin telomere replication and linear plasmid recombination, including antigenic variation, are discussed.     

Changes in streptonigrin lethality during adaptation of Escherichia coli to picolinic acid correlation with intracellular picolinate and iron uptake

Uptake studies with [¹⁴C]picolinate and ⁵⁵Fe³⁺ have provided an explanation for the change in streptonigrin killing on adaptation of Escherichia coli to picolinate, in terms of the available iron within the cell. When picolinic acid is added to a growing culture of E. coli an interval of bacteriostasis ensues; this adaptation period is followed by resumption of exponential growth. Addition of picolinate (4 mM) to a log phase culture of strain W3110 gave protection from the lethal action of streptonigrin (30 μM) when the two agents were added simultaneously. In contrast streptonigrin killed cells that had adapted to picolinate; however, a preincubation of adapted W3110 with phenethyl alcohol protected the cells from streptonigrin lethality. [¹⁴C]Picolinate uptake studies showed that initially picolinate entered the cells, but that it was excluded from adapted cells; addition of phenethyl alcohol permitted the entry of picolinate into adapted W3110. The changes in streptonigrin killing parallel the changes in concentration of intracellular picolinate, which can chelate the iron required by streptonigrin for its bactericidal action. ⁵⁵Fe³⁺ uptake studies showed that initially picolinate prevented iron accumulation by strain W3110, whereas adapted cells did take up iron in the presence of picolinate. Addition of phenethyl alcohol prevented any observed uptake of iron by adapted W3110. This modulation of iron transport by picolinate also affects streptonigrin lethality. Experiments with iron transport mutants showed that picolinate acted on both the enterochelin and citrate routes of uptake. Therefore picolinate affects the concentration of available iron within the cell both by (a) its intracellular presence resulting in chelation of iron and (b) its action on iron uptake; these effects explain the change in streptonigrin killing on adaptation of E. coli to picolinate.     

Delayed disruption of the telomeric links between chromosomes in experimentally induced cells with micronuclei exposed to 5-bromodeoxyuridine in the 1st S period after colcemid administration

Under a long-term administration of colcemid in the Chinese hamster cell culture some cells with micronuclei are seen to form. In the case of co-treatment with colcemid and 5-bromodeoxyuridine (5-BrdU) at metaphases of the first division of cells with micronuclei polycentric chromosomes were observed. These polycentric chromosomes occur due to delayed disruption of telomeric links, previously existing in the interphase. During colcemid treatment the cells pass through two S-periods: one in mononuclear cells, the other in cells with micronuclei. This phenomenon was tested according to the frequency of metaphases with dicentrics after 5-BrdU-treatment of cells at the first or second S-period or during the two cycles of chromosome replication. The 5-BrdU treatment during the first cycle or two cycles of replication resulted in the same frequency of cells with dicentrics--about 50%. The treatment with colcemid alone during two cycles of replication and administration 5-BrdU at the second S-period results in a considerably lower amount (%) of cells with dicentrics--about 10%. Thus, the delayed disruption of telomeric links between chromosomes may occur under the treatment with 5-BrdU at the first S-period after colcemid administration. It is also concluded that this phenomenon can be reproduced in cell with micronuclei when 5-BrdU is incorporated differentially in the sister chromatids.     

Arabidopsis thaliana Chromosome 4 Replicates in Two Phases That Correlate with Chromatin State

DNA replication programs have been studied extensively in yeast and animal systems, where they have been shown to correlate with gene expression and certain epigenetic modifications. Despite the conservation of core DNA replication proteins, little is known about replication programs in plants. We used flow cytometry and tiling microarrays to profile DNA replication of Arabidopsis thaliana chromosome 4 (chr4) during early, mid, and late S phase. Replication profiles for early and mid S phase were similar and encompassed the majority of the euchromatin. Late S phase exhibited a distinctly different profile that includes the remaining euchromatin and essentially all of the heterochromatin. Termination zones were consistent between experiments, allowing us to define 163 putative replicons on chr4 that clustered into larger domains of predominately early or late replication. Early-replicating sequences, especially the initiation zones of early replicons, displayed a pattern of epigenetic modifications specifying an open chromatin conformation. Late replicons, and the termination zones of early replicons, showed an opposite pattern. Histone H3 acetylated on lysine 56 (H3K56ac) was enriched in early replicons, as well as the initiation zones of both early and late replicons. H3K56ac was also associated with expressed genes, but this effect was local whereas replication time correlated with H3K56ac over broad regions. The similarity of the replication profiles for early and mid S phase cells indicates that replication origin activation in euchromatin is stochastic. Replicon organization in Arabidopsis is strongly influenced by epigenetic modifications to histones and DNA. The domain organization of Arabidopsis is more similar to that in Drosophila than that in mammals, which may reflect genome size and complexity. The distinct patterns of association of H3K56ac with gene expression and early replication provide evidence that H3K56ac may be associated with initiation zones and replication origins.     

Din7 and Mhr1 expression levels regulate double-strand-break–induced replication and recombination of mtDNA at ori5 in yeast

The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve homoplasmy, and they also induce homologous recombination to maintain mitochondrial genome integrity. Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown. In Saccharomyces cerevisiae, double-strand breaks (DSBs) introduced by Ntg1 at the mitochondrial replication origin ori5 induce homologous DNA pairing by Mhr1, and reactive oxygen species (ROS) enhance production of DSBs. Here, we show that a mitochondrial nuclease encoded by the nuclear gene DIN7 (DNA damage inducible gene) has 5′-exodeoxyribonuclease activity. Using a small ρ⁻ mtDNA bearing ori5 (hypersuppressive; HS) as a model mtDNA, we revealed that DIN7 is required for ROS-enhanced mtDNA replication and recombination that are both induced at ori5. Din7 overproduction enhanced Mhr1-dependent mtDNA replication and increased the number of residual DSBs at ori5 in HS-ρ⁻ cells and increased deletion mutagenesis at the ori5 region in ρ⁺ cells. However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination. Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5.     

Probing cellular processes with oligo-mediated recombination and using the knowledge gained to optimize recombineering

Recombination with single-strand DNA oligonucleotides (oligos) in Escherichia coli is an efficient and rapid way to modify replicons in vivo. The generation of nucleotide alteration by oligo recombination provides novel assays for studying cellular processes. Single-strand exonucleases inhibit oligo recombination, and recombination is increased by mutating all four known exonucleases. Increasing oligo concentration or adding nonspecific carrier oligo titrates out the exonucleases. In a model for oligo recombination, λ Beta protein anneals the oligo to complementary single-strand DNA at the replication fork. Mismatches are created, and the methyl-directed mismatch repair (MMR) system acts to eliminate the mismatches inhibiting recombination. Three ways to evade MMR through oligo design include, in addition to the desired change (1) a C·C mismatch  6 bp from that change; (2) four or more adjacent mismatches; or (3) mismatches at four or more consecutive wobble positions. The latter proves useful for making high-frequency changes that alter only the target amino acid sequence and even allows modification of essential genes. Efficient uptake of DNA is important for oligo-mediated recombination. Uptake of oligos or plasmids is dependent on media and is 10,000-fold reduced for cells grown in minimal versus rich medium. Genomewide engineering technologies utilizing recombineering will benefit from both optimized recombination frequencies and a greater understanding of how biological processes such as DNA replication and cell division impact recombinants formed at multiple chromosomal loci. Recombination events at multiple loci in individual cells are described here.     

In vitro genetic labeling of Bacillus subtilis cryptic plasmid pHV400.

A DNA segment which encodes resistance to tetracycline, and cannot replicate autonomously, was excised by HindIII endonuclease from plasmid pT127 and joined to the cryptic Bacillus subtilis plasmid pHV400. The analysis of resulting chimerae has allowed us to identify a 1.8 × 10⁶ segment of pHV400 which carried the replication functions of the cryptic plasmid. Another DNA segment, designated pHV32, which can replicate in Escherichia coli but not in B. subtilis has also been used for genetic labeling of the replication region of pHV400. pHV32 is convenient for use in isolating cryptic replicons active in B. subtilis because (1) it can be prepared in large quantities, free from any interferring B. subtilis replicons, from an appropriate E. coli strain; (2) it carries unique sites for various restriction endonucleases; (3) the chloramphenicol resistance gene which it specifies can transform B. subtilis at a high efficiency (10⁶–10⁷ transformants/μg of DNA).     

Replication of bacteriophage lambda DNA dependent on the function of host and viral genes. I. Interaction of red, gam and rec

We have studied the role of the red and gam genes in lambda replication, after infection of wild type and two recombination deficient hosts. Our results show that the rate of phage DNA replication is abnormally low in the absence of red function, in rec⁺ as well as rec^? (A^? and A^?B^?) bacteria. It appears that the virus general recombination proteins play some role in lambda replication that cannot be assumed by the general recombination proteins of its bacterial host. The red^? defect in replication results in a decrease in the total amount of intracellular phage DNA. This DNA, nevertheless, seems normal in structure and is matured and packaged with good efficiency.In rec⁺ and recA^? hosts infected with gam^? mutants, the rate of lambda replication is also low, but in this case, abnormal DNA structures are produced at late times. The gam mutation seems to alter the program of replication such that circular molecules are produced not only at early times, but continuously, throughout the lytic cycle. This, and other facts, suggest that the gam protein is required for the transition from “early” to “late” replication. This requirement for gam function is not observed in recA^?B^? hosts, in which gam mutants replicate at a normal rate and produce DNA indistinguishable from that made by wild type phage. Thus, the gam requirement seems to involve an interaction of this phage protein with the product of the host's recB gene. Other evidence for such interaction comes from our finding that, in vivo, the gam protein does inhibit presumed action of the host's BC nuclease.In the gam^? mutant infections, which are blocked in late replication, absence of a general recombination system seems to create a severe defect in maturation of intracellular phage DNA. This defect, unlike the one affecting λ replication rate, can be alleviated by either the red or rec functions and is correlated with the inability of the mutant phages to make DNA concatemers. Since other late functions (i.e. late messenger RNA production) appear to be normal, we conclude that concatemer formation, via replication or recombination, is an essential step in phage development.     

Asynchronous DNA Synthesis in a Duplicated Chromosomal Region of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

THERE is a highly ordered temporal sequence in the replication of DNA in the polytene chromosomes of Drosophila^1–10. The mechanism underlying this replicative organization remains unknown, but it has been shown that homologous chromosome regions replicate their DNA synchronously whether or not they are paired¹¹ and, in the one case in which it has been studied, this synchrony remains evident even when one of the two homologous regions is translocated to an abnormal position¹². These observations suggest that an essential part of the system controlling replication pattern is located in each of the small chromosome regions, replication of which can be resolved autoradiographically. The simplest model consistent with these assumptions involves a chromosome constituted of numerous “replicons” with replication times geared to a common control mechanism but are independent of the anatomical ordering of the “replicons” within the genome.     

Disappearance of the heteroplasmic state for chloroplast markers in zygospores of Chlamydomonas reinhardtii

In the investigations reported here, the length of zygospore incubation or “maturation” prior to the induction of meiosis was found to affect the inheritance pattern of chloroplast genes. The frequency of zygospores transmitting chloroplast alleles from both parents drops with increasing zygospore age following mating, while the frequencies of zygospores homoplasmic for maternal or paternal chloroplast alleles increase correspondingly. Since there is a negligible reduction in viability, zygospores which are initially biparental appear to become pure for the chloroplast genes from one or the other parent prior to the occurrence of cell division. These results are amplified in crosses of mt⁺ cells which have been irradiated with ultraviolet (uv) light or grown in the presence of the base analog, 5-fluorodeoxyuridine, which also perturbs maternal inheritance. Low doses of uv irradiation, applied to zygospores derived from crosses in which the maternal parent was also irradiated prior to mating, increase the biparental zygospore frequency while reducing the proportion of maternal zygospores. This indicates that at least some maternal zygospore clones are actually derived from zygospores which still contain both parental chloroplast genomes prior to the induction of germination. Thus, a subclass of zygospores must contain paternal chloroplast genomes which are either eliminated upon germination or are not expressed in the resulting zygospore clone. Tetrad analysis of biparental zygospores derived from uv-irradiated mt⁺ gametes demonstrates that the frequency of maternal chloroplast alleles in biparental zygospores decreases as they age. One result is an increase in the proportion of meiotic products homoplasmic for all paternal markers. The increased segregation of homoplasmic daughter cells during the meiotic divisions may result from a reduction in chloroplast ploidy by elimination of maternal genomes. Alternatively, it may reflect an altered ratio of maternal:paternal genomes due to continuous rounds of pairing and gene conversion between heterologous chloroplast DNAs leading to genetic drift within the DNA population of the organelle.     

Characterization of vaccinia virus DNA replication mutants with lesions in the D5 gene

The vaccinia virus D5 gene encodes a 90 kDa early protein that is essential for viral DNA replication. In this report we map and explore the phenotypes of the temperature sensitive mutants bearing lesions in this gene:ts17,ts24,ts69, (WR strain) andts6389 (IHD strain). Viral DNA synthesis was virtually undetectable during non-permissive infections performed withts17, and incorporation of³H-thymidine ceased rapidly when cultures were shifted to the non-permissive temperature in the midst of replication. The D5 protein may therefore be involved in DNA synthesis at the replication fork. The lesions of the four mutants were localized within the D5orf by marker rescue, and the single nucleotide changes responsible for thets phenotype of the three WR mutants were identified. Unexpectedly, the three alleles with N-terminal mutations were impaired in marker rescue when homologous recombination with small (<2 kb), intragenic DNA fragments at 39.5°C was required. This deficiency was not due to degradation of transfected DNA under non-permissive conditions. Efficient marker rescue could be restored by incubation at the permissive temperature for a brief period after transfection, suggesting a requirement for functional D5 in genome/plasmid recombination. Marker rescue under non-permissive conditions could alternatively be restored by co-transfection of unlinked but contiguous DNA sequences.     

Homologous recombination prevents methylation-induced toxicity in Escherichia coli

Methylating agents such as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and methyl methane sulfonate (MMS) produce a wide variety of N- and O-methylated bases in DNA, some of which can block replication fork progression. Homologous recombination is a mechanism by which chromosome replication can proceed despite the presence of lesions. The two major recombination pathways, RecBCD and RecFOR, which repair double-strand breaks (DSBs) and single-strand gaps respectively, are needed to protect against toxicity with the RecBCD system being more important. We find that recombination-deficient cell lines, such as recBCD recF, and ruvC recG, are as sensitive to the cytotoxic effects of MMS and MNNG as the most base excision repair (BER)-deficient (alkA tag) isogenic mutant strain. Recombination and BER-deficient double mutants (alkA tag recBCD) were more sensitive to MNNG and MMS than the single mutants suggesting that homologous recombination and BER play essential independent roles. Cells deleted for the polA (DNA polymerase I) or priA (primosome) genes are as sensitive to MMS and MNNG as alkA tag bacteria. Our results suggest that the mechanism of cytotoxicity by alkylating agents includes the necessity for homologous recombination to repair DSBs and single-strand gaps produced by DNA replication at blocking lesions or single-strand nicks resulting from AP-endonuclease action.     

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.     

Chk1 requirement for high global rates of replication fork progression during normal vertebrate S phase

Chk1 protein kinase maintains replication fork stability in metazoan cells in response to DNA damage and DNA replication inhibitors. Here, we have employed DNA fiber labeling to quantify, for the first time, the extent to which Chk1 maintains global replication fork rates during normal vertebrate S phase. We report that replication fork rates in Chk1^−/− chicken DT40 cells are on average half of those observed with wild-type cells. Similar results were observed if Chk1 was inhibited or depleted in wild-type DT40 cells or HeLa cells by incubation with Chk1 inhibitor or small interfering RNA. In addition, reduced rates of fork extension were observed with permeabilized Chk1^−/− cells in vitro. The requirement for Chk1 for high fork rates during normal S phase was not to suppress promiscuous homologous recombination at replication forks, because inhibition of Chk1 similarly slowed fork progression in XRCC3^−/− DT40 cells. Rather, we observed an increased number of replication fibers in Chk1^−/− cells in which the nascent strand is single-stranded, supporting the idea that slow global fork rates in unperturbed Chk1^−/− cells are associated with the accumulation of aberrant replication fork structures.