J. Herbert Taylor

Increase in DNA replication sites in cells held at the beginning of S phase

CHO cells were pulse labeled with 3H-thymidine after synchronization and blockage at the beginning of S phase for various intervals. The distribution of initiation sites for DNA replication and rates of chain growth were measured in autoradiographs prepared from these cells. Origins used for replication are widely distributed at or near the beginning of S phase, but usable origins increase continuously for many hours when FdU is used to block the synthesis of thymidylate. Potential origins are located about four microns apart, but in normal replication in these fibroblasts only one in 15 to 20 potential origins are used for initiation. On the other hand, when cells are held at the beginning of S phase for 12–14 h, about one-half of the potential origins are activated in part of the DNA and utilized when the cell is released from the block by supplying 3H-thymidine (10−6M). Chain growth during a short pulse decreases with time of the blockage at what appears to be a linear rate. However, cells can replicate long continuous stretches of their DNA with only 2×10−8M thymidine available in the medium for several hours when synthesis is blocked by FdU. The total amount of DNA replicated is, however, much less than when a concentration of 10−6 M thymidine is supplied for the same period. The origins which are finally used under any experimental condition appear to be a random sample of the total potential origins which are distributed in a regular repeating sequence along the DNA at about 12 kilobase intervals.

Early events in the replication and integration of DNA into mammalian chromosomes

Abstract Analysis of DNA produced in pulses of H 3 -thymidine (30 seconds to 2 minutes) indicates that replication of DNA in chromosomes of higher cells proceeds by the production of short segments of perhaps 1000 nucleotides. These segments apparently separate from the template during lysis of cells. However, a large fraction of the DNA remains on the template after a 2-minute pulse. This DNA is linked into chains most of which have sedimentation coefficients in the range of 15–20 S or greater in alkaline sucrose. After a 1-hour chase the new chains have lengths equivalent to the template DNA most of which sediments with a peak at 70–75 S.

Dna Methylation in Eukaryote

The DNA of higher eukaryotes contains one minor base, namely 5-methylcytosine. The distribution of this minor base between different species and different DNA fractions will be considered together with the actual sequences methylated. The properties of the enzyme responsible for DNA modification will be reviewed, particular note being paid to the efficiency of methylation of different DNA substrates. Various possible functions of the 5-methylcytosine in DNA will be considered and particular attention will be paid to the finding that specific modified bases present in DNA not undergoing transcription are absent in the same genes when these are being actively transcribed.

Evidence for a four micron replication unit in CHO cells

CHO cells in culture were synchronized by mitotic selection, allowed to reattach to plastic flasks, and reach S phase in the presence of fluorodeoxyuridine at concentrations known to completely block the synthesis of thymidylate. The cells were released from the block with 3H-thymidine for pulses of 4, 8, 12, 24 and 40 min and DNA fiber autoradiographs prepared. An analysis of the spacing between origins of replication indicates that sites are available at intervals of about 4 μm along most of the DNA. Chain growth proceeds at about 1,000 nucleotides per minute and some of the closely situated sites become continuous, labeled segments after 8–12 min. However, unlabeled segments are still present between the replicated segments after 40 min. The data may be interpreted as evidence for regularly spaced initiation sites which are available in CHO cells, even though only one in 10–15 of these may be utilized for initiation each cycle under normal growth conditions in these cultures.

Studies of bromouracil deoxyriboside substitution in DNA of bean roots (Vicia faba)

Abstract Bean roots were grown in solutions containing BUdR ‡ and an inhibitor of thymidylate synthesis. After various intervals DNA was extracted and analyzed by centrifugation in cesium chloride gradients. DNA of hybrid density increased in amount during the first replication cycle. After some cells had entered the second replication cycle in BUdR, fully substituted DNA appeared as predicted on the basis of semi-conservative replication. However, in all preparations from roots grown in BUdR, a relatively large proportion of the BUdR was incorporated into what appears to be partially substituted chains. The DNA containing this BU banded in a bimodal distribution which indicated a density only a little higher than unsubstituted DNA. After a period of growth in BUdR followed by incorporation of [ 3 H]thymidine, the thymidine was added almost exclusively to the partially substituted fractions of DNA. When limited substitution of BUdR occurred without an inhibitor for thymidylate synthesis, the incorporation was almost exclusively into these bimodally distributed fractions. No hybrid with a fully substituted chain was produced under the latter conditions.

Programmed synthesis of deoxyribonucleic acid during the cell cycle

SummarySynchronization of cells by nearly quantitative removal of late stages of division by mechanical means is possible with a Chinese hamster cell line. By the use of this technique and radioactive and density labels, the cell cycle was shown to be 15 to 16 hr with G1 (5.5 hr), S phase (6.5 hr), and G2 plus division stages 3.5 hr. Bromodeoxyuridine (BUdR), the density label substituted for thymidine, was shown to delay the S phase for about 3 hr, but once the initiation began with BUdR the progress through S phase was about as fast as normal. These experiments were planned to test the hypothesis that all of the deoxyribonucleic acid (DNA) in mammalian cells is highly programmed in temporal sequence of replication so that a replicating unit which is synthesized early in one cycle is invariably replicated early in the next cycle. The data indicate a high degree of temporal programming, but more information is required to be sure how precisely this temporal regulation of replication is controlled and its significance with respect to control of transcription.Synchronization of cells by nearly quantitative removal of late stages of division by mechanical means is possible with a Chinese hamster cell line. By the use of this technique and radioactive and density labels, the cell cycle was shown to be 15 to 16 hr with G1 (5.5 hr), S phase (6.5 hr), and G2 plus division stages 3.5 hr. Bromodeoxyuridine (BUdR), the density label substituted for thymidine, was shown to delay the S phase for about 3 hr, but once the initiation began with BUdR the progress through S phase was about as fast as normal. These experiments were planned to test the hypothesis that all of the deoxyribonucleic acid (DNA) in mammalian cells is highly programmed in temporal sequence of replication so that a replicating unit which is synthesized early in one cycle is invariably replicated early in the next cycle. The data indicate a high degree of temporal programming, but more information is required to be sure how precisely this temporal regulation of replication is controlled and its significance with respect to control of transcription.

Initiation of DNA replication in chromosomes of Chinese hamster ovary cells.

The initiation of DNA replication and the subsequent chain elongation were studied using Chinese hamster ovary cells synchronized at the beginning of S phase. The cells were synchronized by a combination of mitotic selection and treatment with 5-fluorodeoxyuridine (FdU). The use of this drug at a concentration of 10−5 M was found to effectively prevent the leakage of cells into S phase. Reversal of the FdU block by supplying thymidine resulted in the synchronous onset of initiation at multiple sites in each cell. The length of the nascent chains, as determined by autoradiography and velocity sedimentation in alkaline gradients, increased linearly with time during the first twenty minutes of S phase after release. — We applied these procedures to study the effects of the length of an FdU block on the number of functional origins per cell, the rate of chain growth, and the rate of DNA synthesis per cell following reversal of the block. Although no change was noted in the rate of DNA synthesis in cells held at the beginning of S phase from 10.5 to 24 h after division, the rate of chain growth decreased from 0.94 to 0.28 microns per min. This decrease indicated that the number of functional origins increased markedly with length of FdU block. The calculated number of utilized origins per cell increased from 1,900 to 5,700. We also presented arguments that 1,900 origins per cell represents the approximate number of origins utilized by any cell held at the beginning of S phase for less than 10.5 h after division.

Length distributions of single-stranded DNA in Chinese hamster ovary cells.

Abstract The distribution of lengths of single-strand DNA in Chinese hamster ovary cells in the G1 phase of the cell cycle has been observed for various conditions of cell lysis and incubation of the lysates. The method of analysis was band sedimentation through a self-generating density gradient, a technique developed originally for the analytical ultracentrifuge, but modified here for the preparative ultracentrifuge so that measurements of sedimentation coefficients could be made under conditions that minimize shearing of the single-stranded DNA. The effect of rotor speed dependence of the sedimentation coefficient is considered in developing the relation between the sedimentation coefficient and molecular weight for this technique. Special precautions were taken to ensure that complete separation of long single strands took place upon alkaline denaturation, to preclude the possibility of anomalous sedimentation due to interstrand entanglement. Bromodeoxyuridine was incorporated into the DNA in the last round of replication. Advantage was taken of the increased sensitivity to ultraviolet irradiation for the production of single-strand breaks in DNA strands substituted with bromodeoxyuridine. After irradiation the bromodeoxyuridine-substituted strand could be completely separated from the complementary strand in alkaline sedimentation profiles without any apparent breakage in the unsubstituted strand. The conditions of lysis, chosen to minimize the degradation of DNA in the lysates, included lysis at pH 9.3 with Pronase and lysis at high pH (10.8 and 12.0). Sedimentation analysis was performed at various time intervals after incubation at 4 °C or 37 °C. Lysis and incubation at pH 12.0 produced a continuous single-strand breakdown of the DNA in the lysate. Analysis of the sedimentation profiles indicates that these alkaline-induced breaks are randomly distributed. However, lysis and incubation at pH 10.8 and at pH 9.3 with Pronase produced stable sedimentation profiles with number-average molecular weights of 1.7 × 108 and 6.0 × 107, respectively. Analysis of the single-strand DNA sedimentation profiles for these lysates indicates that the distribution of lengths of single-stranded DNA is non-random, i.e. that the distributions may represent regular subunits of chromosomal DNA structure. Suggestive evidence is presented that the approximately 60-μm units are structurally alternated in the two chains. The possible origin of the discontinuities between the subunits is also discussed.

Units of DNA Replication in Chromosomes of Eukaryotes

Publisher Summary This chapter discusses the units of DNA replication in chromosomes of eukaryotes. Considerable evidence has accumulated, which indicates that replication in chromosomes is regulated in a way, such that specific fractions or segments are replicated at specific times in the S phase. The replication of specific segments of chromosomes at limited times in the S phase is indicated by autoradiographic studies involving the X chromosome, the Y chromosome, and selected autosomes of the Chinese hamster genome. The chapter discusses a tentative model, which takes into account what is known about the organization of the genomes of higher cells and their regulatory features.

Origins of replication and gene regulation

SummaryEukaryotic chromosomes appear to consist of many replicons, the time of replication of which is probably controlled by specific origins. However, plasmids without specific eukaryotic origins may also replicate in some cells when injected into nuclei or transferred during transformation. The efficiency and the mechanisms of their initiation are still uncertain. A number of reports are cited which indicate that natural eukaryotic DNAs initiate their replication from specific origins. The nature of these origins are known in only a few instances and no general conclusions can yet be given about the nucleotide sequences involved. Short dispersed repeats of the Alu type appear to function as origins since they enhance the efficiency of replication of vector plasmids in Xenopus eggs. Certain sequences from a variety of eukaryotic DNAs also enhance the replicative potential of plasmids in yeast cells. The common features of such initiators or enhancers is uncertain. If dispersed repeats are origins in mammalian chromosomes, the number appears to be excessive. Either only a subset are functional, or the functional ones are only suborigins in larger replicons in which master origins (not yet isolated) function in the regulation of the timing of replication.Evidence is cited which indicates that the regulation of the time of replication of a gene or gene cluster is part of a regulatory system that makes the DNA available for transcription or leaves it in an inactive state. About one-half the DNA in mammalian cells is replicated in the first half of S phase (SE) After a brief pause in mid-S phase, the remainder of the DNA is replicated in what is designated late S (SL). The fractions replicated in SE and SL may vary in other phylogenetic groups, but wherever division of differentiated cells occurs such fractions are likely to be found. The following hypothesis is proposed. The DNA replicated in SL is suppressed in transcription, if it has the appropriate promoter regions, because the newly replicated DNA is complexed with proteins that suppress transcription. These proteins are only available during SL. Those genes replicated in SE are complexed with a different set of proteins which leave the promoter regions open for transcription when the appropriate regulatory molecules are available. In this way an inactive state or potentially active state can be transmitted from one cell generation to the next.Evidence is cited which indicates that genes which are active in all cells at some stage in the cell cycle are replicated in SE. Other genes which are required in only some tissue or tissues are replicated in SL in all cells except those where the genes are potentially functional. This means that some origins always operate in SE and perhaps some operate only in SL, but other origins can be modified to operate in either SE or SL.The modification may involve methylation of origins since azacytidine has recently been reported to shift the time of replication of segments of chromosomes as well as change genes to a potentially function state by leading to the deletion of cytosine methylation which is normally maintained at specific sites by DNA methyl transferases.