DNA synthesis in polyoma virus infection. II. Relationship between viral DNA replication and initiation of cellular DNA replicons

The rate of synthesis of cellular DNA is stimulated in stationary phase mouse embryo cells infected with polyoma virus. Nascent cellular DNA strands pulselabeled with [³H]thymidine in the presence of replicating viral DNA are smaller, by an average of 2·1 × 10⁷ daltons, than DNA made under similar conditions in uninfected cells. Previous work (Cheevers et al., 1972) has indicated that this observation is the consequence of activation in infected cells of cellular DNA initiation sites not in operation during a similar pulse-labeling interval in uninfected cells. Similar results were obtained using cells infected with the temperature-sensitive Ts-a mutant of polyoma at 32 °C, which permits both the induction of cellular DNA synthesis and replication of viral DNA. However, at a temperature of 39 °C, which permits only the induction of cellular DNA replication in Ts-a-infected cells, the size of newly synthesized DNA is not different from that of uninfected cells. Similarly, in rat embryo cells abortively infected with polyoma (wild-type), stimulation of cellular DNA synthesis occurs but viral DNA replication is restricted, and no difference is apparent in the size of newly formed DNA as compared to uninfected cells. These results are interpreted to mean that in productively infected cells, polyoma DNA and some regions of the host genome may be co-ordinately replicated.     

Preliminary characterization of the temperature-sensitive defect in DNA replication in a mutant mouse L cell

Temperature-sensitive ts A1S9 mouse L cells synthesize DNA apparently normally for 6–8 hr upon incubation at 38.5°C. Thereafter, these cells are able to perform limited polydeoxyribonucleotide chain synthesis at the high temperature, but are unable to convert newly replicated small single-strand segments of DNA (of the order of molecular weight 10⁶ daltons) to large molecular weight chromosomal DNA. Data obtained are compatible with a model which suggests that ts A1S9 cells are able to carry out most individual reactions of DNA synthesis at the high temperature, but are temperature-sensitive in a protein which participates in the joining of small DNA segments to make chromosomal DNA strands. When cells are reincubated at a permissive temperature, after the temperature-sensitive lesion has been established, they recover the latter capability several hours before they are able once again to synthesize DNA at normal rates.     

Effect of Caffeine on DNA Synthesis in Mammalian Cells

Alkaline sucrose sedimentation studies of DNA from mouse lymphoma cells (L5178Y) treated with caffeine have demonstrated the following effects. Caffeine (at a concentration of 1.6 mM) does not introduce strand breaks into preformed DNA nor does it inhibit the rejoining of γ-ray-induced strand breaks. Although it does not affect the over-all rate of DNA synthesis, pulse labeling experiments show that the DNA strands synthesized in its presence are smaller than those made in its absence. This could be the result of (a) DNA being made in shorter replicating units or (b) small gaps in the daughter DNA strands within normal-sized replicating units. These two alternative models were indirectly distinguished as follows. After a pulse label with thymidine-³H in the presence of caffeine, cells were incubated without caffeine in bromodeoxyuridine (BrdUrd). During this incubation, growing strands are elongated and hypothetical gaps (model b) filled in with bromuracil (BrUra)-substituted DNA. The BrUra-containing DNA segments will now be of different lengths on the two models. With smaller replicating units (a) the “elongation segments” will be somewhat smaller than but the same order of magnitude as those in untreated cells, whereas with small gaps (b) the “filled-in gap segments” would be expected to be at least an order of magnitude smaller. The BrUra-containing regions of DNA can be selectively broken open by exposing the cells to light at 313 nm. The exposure required to break open the BUra-substituted regions is inversely related to, and hence gives a measure of, the size of these regions. In caffeine-treated cells these regions were found to be somewhat smaller than but of comparable size with those in untreated cells; this is consistent with the DNA being synthesized in smaller units and argues against the presence of small gaps in the daughter strands.     

Breakage of Parental DNA Strands in Haemophilus influenzae by 313 nm Radiation after Replication in the Presence of 5-Bromodeoxyuridine

Haemophilus influenzae was labeled with thymidine-³H (dThd), then grown in the presence of 5-bromodeoxyuridine (BrdUrd), and then irradiated with 313 nm light (a wavelength that selectively photolyzes DNA containing 5-bromouracil [BrUra]). Irradiation with 313 nm light induced breaks in the ³H-labeled strands in cells grown with BrdUrd at a much higher frequency than in ¹⁴C-labeled DNA of cells not exposed to BrdUrd. Breakage of the ³H-labeled strands was about 0.6% as efficient as that of fully BrUra-substituted DNA. During growth in the presence of BrdUrd, susceptibility to 313 nm-induced breakage of the ³H-labeled DNA strands increased, reaching a maximum in about one generation, and it decreased to zero during subsequent growth for one generation in medium containing dThd instead of BrdUrd. Heat denaturation of DNA extracted from dThd-³H-labeled cells grown in the presence of BrdUrd eliminated 313 nm-induced breakage of the ³H-labeled strands. It is concluded that breakage of the ³H-labeled DNA strands resulted from reaction with photoproducts in the base-paired, BrUra-containing strands, rather than from photolysis of BrdUrd incorporated into parental strands. It may be possible to utilize the phenomenon of interstrand breakage in physical studies of DNA replication.     

The replication of mouse skin epidermal DNA to which is bound 7,12-dimethylbenz(a)anthracene

Experiments were carried out to determine in the intact mouse whether or not mouse skin epidermal DNA to which the polycyclic hydrocarbon DMBA was bound could serve as a template for further DNA replication. Mice which were treated topically with [³H]7,12-dimethylbenz(a)anthracene ([³H]DMBA) received 5-bromodeoxyuridine (BUdR) and 5-fluorouracil (5-FU) in order to incorporate BUdR into replicating DNA which was stimulated to undergo synthesis one or two days later. Epidermal DNA was put on a neutral CsCl gradient and binding of [³H]DMBA was found to both replicated and non-replicated DNA. Separation of the BUdR substituted and non-substituted parental strands of newly replicated DNA an on alkaline CsCl, Cs₂SO₄ gradient showed that the great majority of DMBA was bound to parental strand DNA. The possibility that [³H]DMBA binding was taking place at the same time that labeling with BUdR occurred was eliminated. Thus, these experiments showed that in the intact mouse, skin epidermal DNA to which DMBA is bound can serve as a template for further DNA synthesis.     

Maturation of nascent DNA fragment is disturbed after treatment of cultured mouse FM3A cells with 8-methoxypsoralen plus near-ultraviolet radiation

By the method of sedimentation in 5–20% alkaline sucrose gradient, the process of maturation of the nascent DNA fragment was studied with cultured mouse FM3A cells treated with 8-methoxypsoralen plus near-ultraviolet radiation. This treatment is known to cause crosslinks of the chromosomal DNA strands. The profile of the newly-replicated DNA, labeled for 10 min with [³H]thymidine immediately after treatment, was the same as that of the untreated cells, where the incorporated radioactivity was present in the intermediate DNA fragment (about 50–80 S). But, when the treated cells were labeled after several hours of incubation, the labeled DNA became much shorter due to inhibition of maturation of the initial DNA fragment (the Okazaki fragment) to the intermediate DNA. With the use of aphidicolin, a specific inhibitor of eukaryotic DNA polymerase α, it became apparent that, in addition to formation of the crosslinks, further DNA replication is required to cause this inhibition of DNA maturation. Aphidicolin also suppressed the inhibition of incorporation of [³H]thymidine into cellular DNA after treatment, but inhibition of this incorporation resumed after its removal.     

Hydroxyurea-Induced Accumulation of Short Fragments During Polyoma DNA Replication I. Characterization of Fragments

Hydroxyurea treatment of 3T6 mouse fibroblast cells infected with polyoma virus resulted within 15 min in more than a 20-fold reduction of the rate of both viral and cellular DNA synthesis. After the initial rapid inhibition, the rate of DNA synthesis remained essentially constant for at least 2 h. In the inhibited cells viral DNA accumulated as short chains with a sedimentation coefficient of about 4S (hydroxyurea fragments). A variable proportion of these fragments was released from the template strands when the viral DNA was extracted by the Hirt procedure. Reannealing experiments demonstrated that hydroxyurea fragments were polyoma-specific and probably synthesized on both parental strands at the replication forks.     

DNA Demethylation by DNMT3A and DNMT3B in vitro and of Methylated Episomal DNA in Transiently Transfected Cells

The DNA methylation program in vertebrates is an essential part of the epigenetic regulatory cascade of development, cell differentiation, and progression of diseases including cancer. While the DNA methyltransferases (DNMTs) are responsible for the in vivo conversion of cytosine (C) to methylated cytosine (5mC), demethylation of 5mC on cellular DNA could be accomplished by the combined action of the ten-eleven translocation (TET) enzymes and DNA repair. Surprisingly, the mammalian DNMTs also possess active DNA demethylation activity in vitro in a Ca²⁺- and redox conditions-dependent manner, although little is known about its molecular mechanisms and occurrence in a cellular context. In this study, we have used LC-MS/MS to track down the fate of the methyl group removed from 5mC on DNA by mouse DNMT3B in vitro and found that it becomes covalently linked to the DNA methylation catalytic cysteine of the enzyme. We also show that Ca²⁺ homeostasis-dependent but TET1/TET2/TET3/TDG-independent demethylation of methylated episomal DNA by mouse DNMT3A or DNMT3B can occur in transfected human HEK 293 and mouse embryonic stem (ES) cells. Based on these results, we present a tentative working model of Ca²⁺ and redox conditions-dependent active DNA demethylation by DNMTs. Our study substantiates the potential roles of the vertebrate DNMTs as double-edged swords in DNA methylation-demethylation during Ca²⁺-dependent physiological processes.     

Low-level DNA exchanges in normal human and xeroderma pigmentosum cells after UV irradiation

We have used a T4 endonuclease V assay method for UV-induced pryrimidine dimers in cellular DNA in vivo to obtain evidence for recombinational DNA exchanges after UV irradiation of normal human and Xeroderma pigmentosum (XP) cells. Our data indicate that the endonuclease-sensitive sites in excision-defective XP cells are removed very slowly from the irradiated parental strands and appear concomitantly in daughter strands newly synthesized during post-UV incubation. In the defective XP cells, the extent of appearance of sensitive sites in daughter strands synthesized during a period of 24 h after 10 J/m² appears to be small, probably less than 15% of the initial number of sensitive sites detected in cellular parental strands. Demonstration of such exchanges between normal-density parental and 5-bromodeoxyuridine-labeled daughter strands by alkaline CsCl isopycnic centrifugation was unsuccessful. Further, the extent is much lower in normal human cell because of their efficiet excision repair of the dimers before and after exchanges than in the defective XP cells.     

Replication of polyoma DNA in isolated nuclei. 3. The nucleotide sequence at the RNA-DNA junction of nascent strands

Short fragments consisting of about 100 to 140 deoxyribonucleotides serve as intermediates in the elongation of polyoma DNA. In nuclei isolated from polyoma-infected 3T6 mouse fibroblasts these fragments are initiated by stretches of RNA. We investigated the nature of the ribo- and deoxyribonucleotides at the RNA-DNA link. DNA was synthesized in vitro from each of the four α-³²P-labelled deoxynucleoside triphosphates, the nascent strands were hydrolysed with alkali and the transfer of isotope to ribonucleotides was studied after fractionation of strands according to size. Each strand contained on the average one RNA-DNA link at the 5′ end of DNA. All four common ribo- and deoxyribonucleotides were present at the RNA-DNA link with close to equal frequency, irrespective of chain length or incubation time.In a second approach, daughter strands synthesized in vivo were treated with alkali and the 5′-OH ends of DNA liberated were ³²P-labelled using polynucleotide kinase. All four deoxynucleotides were labelled by this treatment confirming the corresponding results of the in vitro experiments.During the discontinuous synthesis of polyoma DNA the switch from RNA to DNA synthesis is thus not effected by a specific sequence at the RNA-DNA junction, in contrast to Escherichia coli where the sequence p(rPy)p(dC)p was reported.     

Post-replication DNA repair in barley embryos treated with N-methyl-N-nitrosourea

In sterile cultures of free barley embryos, N-methyl-N-nitrosourea (MNU) caused a decrease in the size of both template [¹⁴C]-labeled DNA and of daughter [³H]DNA strands as determined in alkaline sucrose gradients, and inhibited the rate of [³H]thymidine incorporation. In addition, duplexes containing [³H]-daughter DNA analyzed in BND cellulose contained more single-stranded regions in MNU-treated embryos than in the corresponding control. Incubation of MNU-treated embryos in nutrient medium for up to 18 h after the [³H]-labeling permitted the recovery of small-sized daughter DNA to full-sized strands and led to the enhancement of double-strandedness of DNA duplexes containing [³H]-labeled strands. If [³H]-labeling had been carried out 8–10 h after the MNU treatment, the size of daughter DNA, the proportion of double-strandedness and the rate of thymidine uptake into DNA partially increased in comparison with rates observed when labeling had been done just after or 3 h after the MNU treatment, but these variables did not reach the values of the corresponding controls.     

Mechanism of DNA denaturation in the presence of manganese ions

The unwinding of DNA strands in the presence of small concentrations of Mn²⁺ ions (2 × 10^?4?4 × 10^?4M) has been studied. The process of unwinding is nonequilibrium; the DNA strands are gradually unwound at a constant temperature corresponding to the beginning of the melting curve. There is no true renaturation in the partially melted DNA. It is shown in the paper that these effects are due to the aggregation of the unwound DNA regions. The Mn²⁺ ions are responsible for the binding of the unwound strands. The aggregation precludes renaturation, shifts the equilibrium towards the melted state, and causes slow unwinding at a constant temperature. The binding of denaturated regions seems to occur through the guanines.     

On the mechanism of interaction between histone I and DNA and histone III and DNA

A mechanism is suggested at the molecular level whereby histone I can act as a cross-link, or strut, between two DNA strands involved in packing the DNA molecule into the confined space of the chromosome. The amino acid sequence of the N-terminal region is known (to 72). It is suggested that this portion is composed of three main functionally distinct segments: (1) (amino acids 1–18) that forms a broken a-helix (by pro) packed into the depths of the major groove; (2) (amino acids 19–35), rich in lys, which forms a “roof” over segment (1) with nine ionic bonds to phosphate; and (3) (amino acids 41–69) which forms an a-helix. Thus segments (1) and (2) grip the DNA helix and segment (3) forms a strut between two DNA strands. Presumably the rest of the histone molecule forms a second lys-rich “hand” grasping the second DNA helix.The amino acid sequence of histone III suggests that it provides a variant on this thesis. In this case the ionic links to phosphate and the packing of the major groove are provided by successive segments of the protein (e.g. a sequence -arg-lys- followed by a β-turn or a short segment of a-helix). This covers amino acids 1–87. The next segment (88–114) forms an a-helix and the last segment (115–135) repeats the structure of the first segment. Thus histone III might also form cross-links between two parallel DNA strands but its attachment is markedly asymmetrical with one “hand” composed of 87 amino acids filling over $\text{1}\text{1}\text{2}$ turns of the major groove and the other “hand” composed of only 20 amino acids.     

G4 DNA replication: I. Origin of synthesis of the viral and complementary DNA strands

The break in the complementary DNA strand of early G4 replicative form II DNA (RFII) and in the viral DNA strand of late RFII DNA was located using two single cleavage restriction enzymes (EcoRI and PstI) and by limited nick translation of the break using DNA polymerase I and ³²P-labelled deoxyribonucleotides followed by digestion with the restriction enzymes HaeIII and HindII. The break in the complementary DNA strand was unique and in HaeIII Z5 close to the EcoRI cleavage site whereas the break in the viral DNA strand was on the other side of the molecule in HaeIII Z2 approxiately 50% away from the EcoRI cleavage site. Distribution of a short ³H pulse in early G4 replicating intermediates that were synthesising both DNA strands at the same time showed that synthesis of the strands started on opposite sides of the molecule and proceeded in opposite convergent directions, suggesting that initiation of synthesis of the two strands was independent and not unified in a single growing fork.     

Characterization of Simian virus 40 DNA component II during viral DNA replication

Replicating molecules of Simian virus 40 DNA labeled during a short pulse with [³H]thymidine have been fractionated by ultracentrifugation methods and the open circular form (DNA component II) has been characterized. The pulse-labeled DNA component II is a relatively small constituent (1 to 3%) of the pool of replicating molecules. Examination of the circular (18 S) and linear (16 S) strands of DNA component II by alkaline sedimentation and by degradation using exonuclease III of Escherichia coli reveals that the newly synthesized DNA is principally in the linear strand. Cleavage of pulse-labeled DNA component II by an fi⁺, R-factor restriction endonuclease from E. coli demonstrates that the interruption in the pulse-labeled strand is specifically located at the termination point for replication.During a chase period of 20 minutes the amount of DNA component II increases to about 6 to 8% of the total labeled viral DNA. The kinetics of formation of superhelical, DNA component I and disappearance of replicative intermediates are linear during the chase period. After several hours of continuous labeling of replicating viral DNA, the DNA component II pool consists mainly of molecules labeled in both strands with the interruption non-specifically located in either strand. These molecules probably arise by the random introduction of single-strand breaks in newly synthesized DNA component I. During short periods of continuous labeling with [³H]thymidine, the ratio of DNA components I to II increases as a function of the pulse duration. These results support a model for 8V 40 DNA replication in which the open circular form is a precursor of the superhelical form.     

Underwound loops in self-renatured DNA can be diagnostic of inverted duplications and translocated sequences

DNA that contains inverted duplications separated by non-inverted sequences often can form characteristic “underwound loops” when it is denatured and reannealed. An underwound loop is a partially double-stranded, partially denatured segment between the inverted duplications and is produced as follows. During the early stages of the reannealing, intrastrand stem-loop structures form with first-order kinetics when the inverted duplications pair. In a slower second-order reaction, complementary strands (each with a stem-loop) reanneal. The stem-loop structures produce a cruciform in the hybrid. Because of the unpaired sequences in the loop, the cruciform is unstable. It can isomerize to a linear duplex by double-strand exchange of complementary sequences in the stems. This process requires co-ordinated axial rotation of the stems and the flanking duplexes as well as rotation of the loops. If, however, complementary sequences in the loops start to pair, axial rotation is prevented and the stem-loop structures are trapped in a metastable state. The strands of separate, closed rings cannot interwind when they pair. Consequently, the loops observed by electron microscopy have variable patterns of single-stranded denaturation bubbles and duplex segments with both right-handed and left-handed winding.We have used underwound loops to identify a short inverted duplication flanking the γδ recombination sequence of Escherichia coli F factor (isolated on φ80 d₃ilv⁺ transducing phage) and to study DNA from phages Mu and P1 in which the G segments are flanked by inverted duplications. When deproteinized adenovirus-2 DNA was denatured and reannealed, some underwound circles the length of the entire chromosome were observed by electron microscopy. These resulted from the restricted interaction of complementary single-stranded rings generated when pairing of the short inverted terminal duplications closed the ends of single strands. Another type of underwound loop was seen in heteroduplexes containing complementary insertion loops located at different positions in the hybridized strands, such as occurs with P1 cam DNAs. All these underwound structures are similar in appearance to the hybrids formed when topologically separate, complementary single-stranded circles of Colicin E₁ DNA were allowed to anneal.     

Evidence for the attachment of RNA to pulse-labeled DNA in the slime mold, Physarum polycephalum

When $\text{Physarum}\text{polycephalum}$ is pulse-labeled for up to 20 minutes with ³H-thymidine and the shortest labeled DNA strands are partially purified by sedimentation through a neutral aqueous sucrose gradient and then through a formamide-sucrose gradient, these short strands band in Cs₂SO₄ isopycnic density gradients at a density greater than that of bulk single-stranded DNA. Their density is brought partially or nearly completely back to that of single-stranded DNA by hydrolysis with pancreatic RNase A or alkali, respectively. Therefore the dense material attached to the short pulse-labeled DNA strands consists at least partially of RNA.     

Patterns of integration of viral DNA in adenovirus type 2-transformed hamster cells

The patterns of integration of viral DNA in five lines of adenovirus type 2-transformed hamster cells have been investigated. Cell lines HE1 to HE5 were obtained by in vitro transformation of hamster embryo cells by ultraviolet light-inactivated Ad2². In all lines, segments in the central parts of the viral genome are missing. The lines HE1, HE2, HE3, HE4 and HE5 contain 2 to 4, 2 to 4, 6 to 10, about 10, and 2 to 3 genome fragment equivalents per cell, respectively.The patterns of integration in lines HE2 and HE3 are identical; however, the viral genome has been amplified in these cell lines to different extents. This result provides evidence for the post-integrational amplification of inserted viral genomes. It is also conceivable that line HE2 may have undergone losses of integrated Ad2 genomes. The persisting Ad2 genomes in lines HE2 and HE3 have deletions in parts of the EcoRI F and D fragments. The remainders of these fragments are linked to cellular DNA. The termini of the segments of the viral genome have been inverted and linked to each other. This linkage could have occurred via a circular intermediate in integration or via tandemly integrated viral genomes with subsequent deletion events. The linkage of the termini of viral DNA might be mediated by short sequences of cellular DNA.In line HE5, approximately 40% of the Ad2 genome is deleted, and the truncated segments, again comprising the terminal Ad2 DNA fragments, have been fused. The termini of the viral DNA are linked to cellular DNA. In lines HE1 and HE4 complex deletion and fusion events have altered the inserted Ad2 genomes.