The presence of single-stranded regions in mammalian DNA

Benzoylated naphthoylated DEAE-cellulose chromatography has been used in this study to demonstrate the presence of single-stranded regions in the DNA of mouse L cells and Chinese hamster ovary cells. In mouse L cell DNA the majority of the single-stranded regions were found to have free ends, as judged from their susceptibility to S₁ endonuclease and exonuclease VII. They were separated on average by a distance of 70 μm.The single-stranded regions fell into two classes, distinguished by whether or not they were derived from the replication fork. Pulse-label administered to Chinese hamster ovary cells was found to be closely associated with the single-stranded regions, as shearing the DNA left a greater proportion of the pulse-label than the bulk-label in the single-strand-containing fraction. However, reasonable estimates of the number of replication forks present at one time showed that the single-stranded regions occurred too frequently along the DNA for them all to be associated with replication. When DNA was isolated from Chinese hamster ovary cells synchronized in G₁ phase, it too was found to contain single-stranded regions, separated by a distance of 100 μm. The regions in the G₁ cells were demonstrated not to be the result of a low level of DNA replication nor to be an artifact of the isolation procedure. Possible functions of this non-replicative class of single-stranded regions are discussed. They may, for example, act as regulatory signals or to be pre-formed initiation points for DNA replication. They are the subject of ongoing investigation.     

Dynamics of association of origins of DNA replication with the nuclear matrix during the cell cycle

DNA of replication foci attached to the nuclear matrix was isolated from Chinese hamster ovary cells and human HeLa cells synchronized at different stages of the G₁ and S phases of the cell cycle. The abundance of sequences from dihydrofolate reductase ori-β and the β-globin replicator was determined in matrix-attached DNA. The results show that matrix-attached DNA isolated from cells in late G₁ phase was enriched in origin sequences in comparison with matrix-attached DNA from early G₁ phase cells. The concentration of the early firing ori-β in DNA attached to the matrix decreased in early S phase, while the late firing β-globin origin remained attached until late S phase. We conclude that replication origins associate with the nuclear matrix in late G₁ phase and dissociate after initiation of DNA replication in S phase.     

Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system

Initiation of DNA replication is tightly controlled during the cell cycle to maintain genome integrity. In order to directly study this control we have previously established a cell-free system from human cells that initiates semi-conservative DNA replication. Template nuclei are isolated from cells synchronized in late G₁ phase by mimosine. We have now used DNA combing to investigate initiation and further progression of DNA replication forks in this human in vitro system at single molecule level. We obtained direct evidence for bidirectional initiation of divergently moving replication forks in vitro. We assessed quantitatively replication fork initiation patterns, fork movement rates and overall fork density. Individual replication forks progress at highly heterogeneous rates (304 ± 162 bp/min) and the two forks emanating from a single origin progress independently from each other. Fork progression rates also change at the single fork level, suggesting that replication fork stalling occurs. DNA combing provides a powerful approach to analyse dynamics of human DNA replication in vitro.     

Replication intermediates formed during initiation of DNA synthesis in methotrexate-resistant CHOC 400 cells are enriched for sequences derived from a specific, amplified restriction fragment

1-beta-D-Arabinofuranosylcytosine (ara-C) inhibits nuclear DNA replication in Chinese hamster ovary cells by an efficient chain termination mechanism without affecting the rate at which cells traverse G1 and enter S [Heintz, N. H., & Hamlin, J. L. (1983) Biochemistry 22, 3557-3562]. Here we have employed ara-C to enrich for replication intermediates formed during initiation of DNA synthesis in synchronized CHOC 400 cells, a methotrexate-resistant derivative of Chinese hamster ovary cells that contains approximately 1000 copies of an early replicating 150-kb chromosomal domain. This highly amplified domain includes the gene for dihydrofolate reductase (DHFR). CHOC 400 cells were collected at the G1/S boundary of the cell cycle with aphidicolin prior to release into S in the presence of both [methyl-3H] thymidine and various concentrations of ara-C. Chromatographic fractionation of restriction endonuclease digests over benzoylated naphthoylated DEAE-cellulose (BND-cellulose) showed that high concentrations of ara-C inhibited the maturation of chromosomal replication intermediates containing ssDNA (replication forks) into dsDNA for up to 60 min. The effect of ara-C on the sequence complexity of replication intermediates formed during early S phase was determined by hybridizing purified intermediates labeled with 32P in vitro to Southern blots of genomic DNA derived from both methotrexate-sensitive and methotrexate-resistant Chinese hamster ovary cells. In the absence of ara-C, 32P-labeled ssDNA BND-cellulose fractions from cultures released into S for 30-60 min hybridized to a spectrum of restriction fragments encompassing 40-50 kb of the amplified DHFR domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Replication of the rRNA and legumin genes in synchronized root cells of pea (Pisum sativum): evidence for transient EcoR I sites in replicating rRNA genes

Summary The temporal pattern of replication of the rRNA and legumin genes differs in synchronized pea root cells. The relative number of rRNA genes replicated hourly during the first five hours of S phase ranges between 5 and 10 percent. In late S phase, during hours six through nine, the number of rRNA genes replicated increases reaching a maximum of about 25 percent at the ninth hour. Unlike the rRNA genes, the legumin genes have a wave-like pattern of replication peaking in early S phase at the third hour and again in late S phase at the eighth hour.Replicating rDNA, isolated by benzoylated naphthoylated DEAE-column chromatography, has EcoR I restriction sites that are absent in non-replicating rDNA sequences. The cleavage of these sites is independent of the time of rDNA replication. The transient nature of the EcoR I sites suggests that they exist in a hemimethylated state in parental DNA.The two Hind III repeat-size classes of rDNA of var. Alaska peas are replicated simultaneously as cells progress through S phase. Thus, even if the 9.0 kb and 8.6 kb repeat classes are located on different chromosomes, their temporal order of replication is the same.     

Murine coronavirus replication induces cell cycle arrest in G0/G1 phase

Mouse hepatitis virus (MHV) replication in actively growing DBT and 17Cl-1 cells resulted in the inhibition of host cellular DNA synthesis and the accumulation of infected cells in the G₀/G₁ phase of the cell cycle. UV-irradiated MHV failed to inhibit host cellular DNA synthesis. MHV infection in quiescent 17Cl-1 cells that had been synchronized in the G₀ phase by serum deprivation prevented infected cells from entering the S phase after serum stimulation. MHV replication inhibited hyperphosphorylation of the retinoblastoma protein (pRb), the event that is necessary for cell cycle progression through late G₁ and into the S phase. While the amounts of the cellular cyclin-dependent kinase (Cdk) inhibitors p21^Cip1, p27^Kip1, and p16^INK4a did not change in infected cells, MHV infection in asynchronous cultures induced a clear reduction in the amounts of Cdk4 and G₁ cyclins (cyclins D1, D2, D3, and E) in both DBT and 17Cl-1 cells and a reduction in Cdk6 levels in 17Cl-1 cells. Infection also resulted in a decrease in Cdk2 activity in both cell lines. MHV infection in quiescent 17Cl-1 cells prevented normal increases in Cdk4, Cdk6, cyclin D1, and cyclin D3 levels after serum stimulation. The amounts of cyclin D2 and cyclin E were not increased significantly after serum stimulation in mock-infected cells, whereas they were decreased in MHV-infected cells, suggesting the possibility that MHV infection may induce cyclin D2 and cyclin E degradation. Our data suggested that a reduction in the amounts of G₁ cyclin-Cdk complexes in MHV-infected cells led to a reduction in Cdk activities and insufficient hyperphosphorylation of pRb, resulting in inhibition of the cell cycle in the G₀/G₁ phase.     

Location of the replication origin in the 9-kb repeat size class of rDNA in pea (Pisum sativum)

The replication origin of the 9-kb rDNA repeat size class of pea (Pisum sativum cv. Alaska) was identified by benzoylated naphthoylated DEAE-cellulose column chromatography and Southern blotting procedures. The origin is located at or near a 0.19-kb EcoR I fragment in the non-transcribed spacer region between the 25S and 18S rRNA genes. Identification of the origin was based on three criteria: (i) an enrichment of the 0.19-kb fragment in replicating rDNA from asynchronously dividing root meristematic cells, (ii) the scarcity of the 0.19-kb fragment in rDNA from non-dividing carbohydrate starved cells, and (iii) a 60-min periodic enrichment of the 0.19-kb fragment in replicating rDNA that temporally coincides with the sequential initiation of replication of replicon families in synchronized pea root cells.     

Radiosensitivity of Mammalian Cells: IV. Effects of X-Irradiation on the DNA Synthetic Period in Synchronized Cells

The effect of X-irradiation on the timing of DNA synthesis in the Chinese hamster ovary cells has been investigated. Mitotically synchronized cells irradiated in mitosis or early G₁ exhibited a fixed, dose-independent (150-2000 rad) delay of 1.6 hr in entry into S, while the duration of S was unaffected. Cells irradiated during late G₁ or the first 0.8 hr of S were not affected either in time of initiation or duration of S. However, when cells 0.8 hr or more into S were irradiated, completion but not initiation of DNA synthesis was delayed, indicating a very precise separation of X-ray effects upon initiation and replication. There was no indication of a re-ordering of cells following irradiation and recovery, since cells in G₂ at the time of irradiation always divided before cells irradiated in S. The results suggest that two separate functions required for initiation and continued replication of DNA may be differentially sensitive to X-irradiation.     

Mimosine arrests proliferating human cells before onset of DNA replication in a dose-dependent manner

The synchronization effects of the plant amino acid mimosine on proliferating higher eukaryotic cells are still controversial. Here, I show that 0.5 mM mimosine can induce a cell cycle arrest of human somatic cells in late G1 phase, before establishment of active DNA replication forks. The DNA content of nuclei isolated from mimosine-treated cells was determined by flow cytometry. The presence or absence of DNA replication forks in these isolated nuclei was then detected by DNA replication run-on assays in vitro. Treatment of asynchronously proliferating HeLa or EJ30 cells for 24 h with 0.5 mM mimosine resulted in a population synchronized in late G1 phase. S phase entry was inhibited by 0.5 mM mimosine in cells released from a block in mitosis or from quiescence. When added to early S phase cells, 0.5 mM mimosine did not prevent S phase transit, but delayed progression through late stages of S phase after a lag of 4 h, eventually resulting in a G1 phase population by preventing entry into the subsequent S phase. In contrast, lower concentrations of mimosine (0.1-0.2 mM) failed to prevent S phase entry, resulting in cells containing active DNA replication foci. The G1 phase arrest by 0.5 mM mimosine was reversible upon mimosine withdrawal. This synchronization protocol using 0.5 mM mimosine can be exploited for studying the initiation of human DNA replication in vitro.     

Site-specific and temporally controlled initiation of DNA replication in a human cell-free system

We have recently established a cell-free system from human cells that initiates semi-conservative DNA replication in nuclei isolated from cells which are synchronised in late G₁ phase of the cell division cycle. We now investigate origin specificity of initiation using this system. New DNA replication foci are established upon incubation of late G₁ phase nuclei in a cytosolic extract from proliferating human cells. The intranuclear sites of replication foci initiated in vitro coincide with the sites of earliest replicating DNA sequences, where DNA replication had been initiated in these nuclei in vivo upon entry into S phase of the previous cell cycle. In contrast, intranuclear sites that replicate later in S phase in vivo do not initiate in vitro. DNA replication initiates in this cell-free system site-specifically at the lamin B2 DNA replication origin, which is also activated in vivo upon release of mimosine-arrested late G₁ phase cells into early S phase. In contrast, in the later replicating ribosomal DNA locus (rDNA) we neither detected replicating rDNA in the human in vitro initiation system nor upon entry of intact mimosine-arrested cells into S phase in vivo. As a control, replicating rDNA was detected in vivo after progression into mid S phase. These data indicate that early origin activity is faithfully recapitulated in the in vitro system and that late origins are not activated under these conditions, suggesting that early and late origins may be subject to different mechanisms of control.     

Cell Cycle-Dependent Regulation of Human DNA Polymerase α-Primase Activity by Phosphorylation

DNA polymerase α-primase is known to be phosphorylated in human and yeast cells in a cell cycle-dependent manner on the p180 and p68 subunits. Here we show that phosphorylation of purified human DNA polymerase α-primase by purified cyclin A/cdk2 in vitro reduced its ability to initiate simian virus 40 (SV40) DNA replication in vitro, while phosphorylation by cyclin E/cdk2 stimulated its initiation activity. Tryptic phosphopeptide mapping revealed a family of p68 peptides that was modified well by cyclin A/cdk2 and poorly by cyclin E/cdk2. The p180 phosphopeptides were identical with both kinases. By mass spectrometry, the p68 peptide family was identified as residues 141 to 160. Cyclin A/cdk2- and cyclin A/cdc2-modified p68 also displayed a phosphorylation-dependent shift to slower electrophoretic mobility. Mutation of the four putative phosphorylation sites within p68 peptide residues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the inhibition of replication activity. Phosphopeptide maps of the p68 subunit of DNA polymerase α-primase from human cells, synchronized and labeled in G₁/S and in G₂, revealed a cyclin E/cdk2-like pattern in G₁/S and a cyclin A/cdk2-like pattern in G₂. The slower-electrophoretic-mobility form of p68 was absent in human cells in G₁/S and appeared as the cells entered G₂/M. Consistent with this, the ability of DNA polymerase α-primase isolated from synchronized human cells to initiate SV40 replication was maximal in G₁/S, decreased as the cells completed S phase, and reached a minimum in G₂/M. These results suggest that the replication activity of DNA polymerase α-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.     

Factors affecting the formation of phytol and its incorporation into chlorophyll by homogenates of the leaves of the french bean Phaseolus vulgaris

The biosynthesis and phosphorylation of histone fractions were measured in synchronized CHO Chinese hamster cells arrested in late G₁ by hydroxyurea treatment. Hydroxyurea was found to inhibit the initiation of both DNA and histone synthesis, thus confirming the conclusion that it arrests cells in G₁ slightly before the $\text{G}{}_1\text{S}$ boundary. However, hydroxyurea did not inhibit the phosphorylation of histone f1 or histone f2a2. The phosphorylation of histone f1, which normally is absent in early G₁, begins 2 hr prior to DNA synthesis. In the presence of hydroxyurea, f1 phosphorylation occurs on schedule at this same time in G₁, resulting in significant G₁-phase f1 phosphorylation. This offers strong evidence that (a) f1 phosphorylation is not restricted to S phase; (b) “old” f1 which was synthesized in previous cell cycles is phosphorylated in G₁ before “new” f1 which is synthesized in S phase; and (c) G₁-phase f1 phosphorylation does not require new histone or new DNA synthesis.Histone f1 phosphorylation was observed to occur at accelerated rates in S phase over phosphorylation rates observed in late G₁-arrest. Data support the proposal that three different levels of f1 phosphorylation occur during the cell cycle: (1) a G₁-related phosphorylation of “old” f1; (2) an S-related phosphorylation of both “old” and “new” f1; and (3) a superphosphorylation of f1 associated with chromosome condensation during the G₂ to M transition. It is also possible that a limited proportion of f1 may be phosphorylated in G₁, perhaps at the initial DNA synthesis sites, and that an increased proportion of f1 is phosphorylated in S as DNA is synthesized. Similarities between the kinetics of histone f1 phosphorylation and the association of DNA with lipoprotein in synchronized control and hydroxyurea-treated cells suggest an involvement of f1 phosphorylation in cell-cycle-dependent chromatin structural changes.     

Histone phosphorylation in late interphase and mitosis

Histone phosphorylation in late interphase has been investigated employing cells synchronized by the isoleucine-deprivation method, followed by resynchronization at the $\text{G}{}_1\text{S}$ boundary using hydroxyurea. Phosphorylation occurred in both f1 and f2a2 as cells synchronously entered S phase following removal of hydroxyurea. The relative rates of phosphorylation of both species of histone increased in G₂-rich and metaphase-rich cultures. A small amount of histone f3 phosphorylation was also observed in M-rich cultures which was not seen in G₁, S, or G₂-rich cultures. It is concluded that f1 phosphorylation is not dependent on continous DNA replication. These experiments suggest consideration of the concept that f1 phosphorylation is initiated as a preparation for impending cell division.     

Calmodulin and cell proliferation

The calmodulin content of synchronized Chinese hamster ovary (CHO-K₁) cells was determined at each phase of the cell cycle. The calmodulin content was minimum in the G₁ phase, increased after the cells entered S phase and reached the maximum level at the late G₂ or early M phase. When 30 μM of W-7 (calmodulin antagonist) was added at the S phase, the cell cycle was blocked at the late G₂ or early M phase. The addition of W-7 also prevented the morphological changes caused by cholera toxin. These results suggest that calmodulin plays an important role in the phases through S to M, possibly in the initiation of DNA synthesis and in the mitosis.     

CIRCADIAN RHYTHMS IN MOUSE EPIDERMAL BASAL CELL PROLIFERATION

Several kinetic parameters of basal cell proliferation in hairless mouse epidermis were studied, and all parameters clearly showed circadian fluctuations during two successive 24 hr periods. Mitotic indices and the mitotic rate were studied in histological sections; the proportions of cells with S and G₂ phase DNA content were measured by flow cytometry of isolated basal cells, and the [³H]TdR labelling indices and grain densities were determined by autoradiography in smears from basal cell suspensions. The influx and efflux of cells from each cell cycle phase were calculated from sinusoidal curves adapted to the cell kinetic findings and the phase durations were determined. A peak of cells in S phase was observed around midnight, and a cohort of partially synchronized cells passed from the S phase to the G₂ phase and traversed the G₂ phase and mitosis in the early morning. The fluctuations in the influx of cells into the S phase were small compared with the variations in efflux from the S phase and the flux through the subsequent cell cycle phases. The resulting delay in cell cycle traverse through S phase before midnight could well account for the accumulation of cells in S phase and, therefore, also the subsequent partial synchrony of cell cycle traverse through the G₂ phase and mitosis. Circadian variations in the duration of the S phase, the G₂ phase and mitosis were clearly demonstrated.     

Human Parvovirus B19 Infection Causes Cell Cycle Arrest of Human Erythroid Progenitors at Late S Phase That Favors Viral DNA Replication

Human parvovirus B19 (B19V) infection has a unique tropism to human erythroid progenitor cells (EPCs) in human bone marrow and the fetal liver. It has been reported that both B19V infection and expression of the large nonstructural protein NS1 arrested EPCs at a cell cycle status with a 4 N DNA content, which was previously claimed to be “G₂/M arrest.” However, a B19V mutant infectious DNA (M20^mTAD2) replicated well in B19V-semipermissive UT7/Epo-S1 cells but did not induce G₂/M arrest (S. Lou, Y. Luo, F. Cheng, Q. Huang, W. Shen, S. Kleiboeker, J. F. Tisdale, Z. Liu, and J. Qiu, J. Virol. 86:10748–10758, 2012). To further characterize cell cycle arrest during B19V infection of EPCs, we analyzed the cell cycle change using 5-bromo-2′-deoxyuridine (BrdU) pulse-labeling and DAPI (4′,6-diamidino-2-phenylindole) staining, which precisely establishes the cell cycle pattern based on both cellular DNA replication and nuclear DNA content. We found that although both B19V NS1 transduction and infection immediately arrested cells at a status of 4 N DNA content, B19V-infected 4 N cells still incorporated BrdU, indicating active DNA synthesis. Notably, the BrdU incorporation was caused neither by viral DNA replication nor by cellular DNA repair that could be initiated by B19V infection-induced cellular DNA damage. Moreover, several S phase regulators were abundantly expressed and colocalized within the B19V replication centers. More importantly, replication of the B19V wild-type infectious DNA, as well as the M20^mTAD2 mutant, arrested cells at S phase. Taken together, our results confirmed that B19V infection triggers late S phase arrest, which presumably provides cellular S phase factors for viral DNA replication.     

An evolving tail of centromere histone variant CENP-A

We have recently shown that replication forks pause near origins in normal human fibroblasts (NHF1-hTERT) but not glioblastoma T98G cells. This observation led us to question whether other differences in the replication program may exist between these cell types that may relate to their genetic integrity. To identify differences, we detected immunoflourescently the sequential incorporation of the nucleotide analogs IdU and CldU into replicating DNA at the start of every hour of a synchronized S phase. We then characterized the patterns of labeled replicating DNA tracks and quantified the percentages and lengths of the tracks found at these hourly intervals. From the directionality of labeling in single extended replicating DNA fibers, tracks were categorized as single bidirectional origins, unidirectional elongations, clusters of origins firing in tandem, or merging forks (terminations). Our analysis showed that the start of S phase is enriched in single bidirectional origins in NHF1-hTERT cells, followed by an increase in clustering during mid S phase and an increase in merging forks during late S phase. Early S phase in T98G cells also largely consisted of single bidirectional origin initiations; however, an increase in clustering was delayed until an hour later, and clusters were shorter in mid/late S phase than in NHF1-hTERT cells. The spike in merging forks also did not occur until an hour later in T98G cells. Our observations suggest models to explain the temporal replication of single and clustered origins, and suggest differences in the replication program in a normal and cancer cell line.     

Polyamine biosynthesis and accumulation during the G1 to S phase transition

Ornithine decarboxylase, an important enzyme in growth regulation, is increased in CHO cells in G₁ phase of the cell cycle and decreases as the cells progress into S phase. S-adenosyl-L-methionine decarboxylase activity, which is dependent on either the presence of putrescine or spermidine for the synthesis of spermidine and spermine respectively, shows a maximal increase in late G₁/early S phase which corresponds very closely with the cell cycle phase specific accumulation of spermidine and spermine during S phase. Total culture evaluation of spermidine and spermine, which included extracellular as well as intracellular concentrations, indicated that extracellular accumulations of these polyamines occurred only in G₁ and that entry into S phase was concomitant with intracellular accumulation patterns. Hyperthermia (43°C for 1 hour) in mid-G₁ phase of the cell cycle resulted in rapid decreases in the activities of ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase. In these cells, DNA replication was also not detectable until nine hours after mitosis, a time at which there had been recovery of ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase activities. Previous data have further indicated a requirement for polyamine reaccumulation before control DNA replication rates are resumed. We therefore suggest that polyamine biosynthesis and intracellular accumulation are both temporal and quantitative prerequisites for transition through S phase.     

Image analysis of in situ cell cycle related changes of PCNA and Ki-67 proliferating antigen expression

Abstract. This study reports on the proliferating cell nuclear antigen (PCNA) and Ki-67 cell cycle related expression and distribution pattern analysed in the same cells. MCF-7 cells were synchronized by mitotic detachment and triple stained for DNA, PCNA and Ki-67. The major cell type was identified on each time sample as a function of the PCNA/Ki-67 pattern, and both antigens as well as DNA were quantified. During G₁ phase, the expression of PCNA greatly increased whereas Ki-67 content decreased. During S phase, nuclear Ki-67 content continuously increased especially in the second half of this phase, mainly due to the accumulation of the antigen in the nucleoli. During G₂ phase, the antigen significantly passed into the nucleoplasm, its content continued to increase and reached its maximum in mitotic cells. Nuclear PCNA content mostly increased in the first part of S phase and sharply declined in mitotic cells as the antigen shifted to the cytoplasm. Cells showing PCNA positive Ki-67 negative labelling were observed in all time samples from the beginning of the experiment. Their nuclear size, DNA content (of G₁ cells), PCNA content (equivalent to the content of some late G, cells) and time occurrence (their percentage increased after the last late G₁ cells had disappeared) tend to indicate that these cells have left the cycle by the end of G₁ phase to enter a quiescent state. Cells coming out of mitosis split into two groups according to their Ki-67/PCNA content. The biggest fraction was PCNA negative and Ki-67 positive while the smallest showed positive staining for both antibodies. Cells of this second cohort slowly lost their 1–67 while their PCNA content increased as they moved through G₁. Concurrently, most of the cells of the first cohort (here called Q2 and Q3 cell types) lost their Ki-67 without increasing their PCNA content; then they joined cells of the second cohort by increasing their PCNA content at the end of G, phase. Some cells of this first cohort can also increase their PCNA and thus reach cells of the first cohort before the end of G₁ phase. The existence of these two main cell cohorts suggests that cells after mitosis differ in some way that make them progress dlfferently through G₁. Some cells seem to go through early G₁ (G_1a and late G₁ (G_lb) while others may come out of mitosis committed to go through the following cycle by directly entering late G₁ compartment.