Karl G. Lark

Induction of replication by thymine starvation at the chromosome origin in Escherichia coli

The sequential replication of the DNA of Escherichia coli 15T − (557) observed during exponential growth was compared with that observed after thymine starvation. Thymine starvation was shown to alter the sequence, initiating replication prematurely at the chromosome origin, i.e. that point on the chromosome at which replication commences after a period of amino acid starvation. After thymine starvation most cells will replicate the chromosome simultaneously from two points—the origin and the growth point present prior to starvation. This abnormal situation per se is not lethal and the replication sequence returns to normal after about one generation. The mechanism of recovery has not been determined. The results show that amino acid starvation prevents premature initiation of DNA synthesis at the origin rather than a terminal step necessary to complete the chromosome.

Segregation of Sister Chromatids in Mammalian Cells

Segregation of sister chromatids in embryonic mouse cells in primary tissue culture is not random. In mitosis those chromatids replicated on a DNA template synthesized during the preceding division cycle are separated from those constructed on a template synthesized two division cycles previously. Segregation in cells of the Chinese hamster follows a similar, but less pronounced, pattern.

Evidence for two distinct aspects of the mechanism regulating chromosome replication in Escherichia coli

The premature initiation of DNA synthesis which occurs in Escherichia coli 15 T-(557) after thymine deprivation can be prevented by fiuorouracil and chloramphenicol, neither of which prevents reinitiation of DNA synthesis following amino acid starvation. The results support the hypothesis that there are at least two proteins which regulate DNA synthesis in bacteria. One of these, postulated to be a structural protein, does not accumulate in the absence of DNA replication.

Initiation of DNA replication in Escherichia coli 15T−: Chronological dissection of three physiological processes required for initiation☆

Abstract The replicative origin of the Escherichia coli chromosome was labeled with [ 3 H]thymine after amino acid starvation. Replication of this label was measured by further growth in [ 13 C]glucose- 15 NH 4 Cl. Such replication served as a measure of the initiation of a new cycle of chromosome replication. A new replication cycle was initiated about 35 minutes after labeling the chromosome origin. Initiation was heterogeneous, occurring earlier in some cells than in others. Two synthetic processes were distinguished. One requires amino acids, and is inhibited by 150 μg chloramphenicol/ml. and by 0.25% phenethyl alcohol. It was not inhibited by 25 μg chloramphenicol/ml. This process was completed about 15 minutes before the actual re-initiation of replication. The other was inhibited by 25 μg of chloramphenicol/ml. and was completed 30 minutes before re-initiation occurred. A third requirement for initiation was suggested by the fact that initiation did not occur immediately after necessary protein synthesis was completed. Both of these processes limit stoichiometrically the initiation of replication since they must be repeated to allow initiation of a second cycle of replication. In the absence of thymine, cells acquired the ability to initiate a new replication cycle despite subsequent inhibition of protein synthesis. The time at which this potential was achieved was independent of DNA synthesis.

Regulation of chromosome replication in Escherichia coli: a comparison of the effects of phenethyl alcohol treatment with those of amino acid starvation☆

Escherichia coli 15T− was treated with phenethyl alcohol at a concentration sufficient eventually to stop DNA synthesis, but not RNA or protein synthesis. DNA synthesis was found to cease at that same region of the chromosome at which it ceases in the absence of required amino acids. It was concluded that phenethyl alcohol allows the completion of a chromosome replication cycle, but prevents the initiation of a new replication cycle. In the presence of phenethyl alcohol, an excess of protein is synthesized which participates in initiating new cycles of DNA replication. Synthesis of this protein is inhibited by chloramphenicol. Although this protein may be necessary for initiating a cycle of DNA replication, it is not sufficient.

DNA replication in Escherichia coli: Replication in absence of protein synthesis after replication inhibition

In Escherichia coli, DNA replication can occur in the absence of protein synthesis after a period of replication inhibition. Such replication will occur after thymine starvation, nalidixic acid treatment or exposure of a DNA temperature-sensitive mutant to a period at the non-permissive temperature. In all cases, subsequent DNA replication continued in the absemce of protein synthesis for many hours. This DNA synthesis could be observed only under conditions in which the restrictive breakdown of unmethylated cellular DNA is prevented. DNA-DNA hybridization experiments indicated that the entire bacterial genome is replicated. It was also found that DNA is synthesized sequentially and that only a portion of the chromosomes are involved in replication at any time. These appear to be chosen at random from the accumulating chromosome pool. Once acquired, this ability to synthesize DNA in the absence of protein synthesis was stable and could be maintained during growth in a complete medium which allowed synthesis and cell division. The ability to replicate DNA in the absence of protein synthesis appears to involve some normal component of the replication apparatus, since conditional lethal mutants exist which are unable to synthesize DNA at a non-permissive temperature and are altered in the ability to synthesize DNA in chloramphenicol after thymine starvation. The mechanism of stabilization of DNA replication which follows the inhibition of DNA synthesis is discussed in relation to the normal initiation and replication of the E. coli chromosome.

Regulation of chromosome replication in Escherichia coli: Alternate replication of two chromosomes at slow growth rates

Escherichia coli 15 T − , arginine − , methionine − , tryptophan − , grows exponentially with a 40-minute generation period in M9-glucose medium and a 70-minute generation period in M9-succinate medium. The DNA content per cell in these two media is 8·4 and 7·0 × 10 9 daltons, respectively, corresponding to about two chromosomes per cell. Autoradiographic studies demonstrate that, at any instant, cells in glucose cultures are synthesizing about twice as many conserved DNA units as cells in succinate cultures. When starved of amino acids, cells growing in glucose synthesize about 2·5 times as much DNA as cells growing in succinate. Experiments combining a density and a radioactive label demonstrated that in succinate the two chromosomes per cell are synthesized alternately and in sequence. In the absence of amino acids, replication of one chromosome is completed. Replication of the second is initiated upon re-addition of amino acids. Thymine starvation initiates replication prematurely on only those chromosomes which are already in the process of replication. A model for alternate chromosome replication is discussed.

Chromosome replication in Escherichia coli 15T− at different growth rates: Rate of replication of the chromosome and the rate of formation of small pieces☆☆☆

Abstract The initiation and termination of chromosome replication in Escherichia coli 15T − have been measured at slow growth rates and after transfer from poor to rich media. Replication of the chromosome (travel-time) requires 80 minutes in aspartate medium (cell doubling time, 120 min). After transfer to glucose (travel-time, 40 min) the rate of replication increases slightly. After transfer to Casamino acids medium (doubling-time, 27 min) a somewhat decreased rate of travel of the replication fork is observed for 50 to 60 minutes, after which it increases to the travel-time characteristic for Casamino acids (40 min). At 37 °C and in all growth media, short pulses of [ 3 H]thymidine will label DNA which can be extracted by alkali in the form of low molecular weight “Okazaki” pieces. These pieces are about 1 μ in length and correspond in number to the replication of 2.5 μ of the chromosome in glucose or Casamino acids medium and 1.5 μ in aspartate medium. The time required to synthesize such a piece is less than 12 seconds and more than 6 seconds at 20 °C. This time is the same in aspartate and glucose media, and after a shift-up from aspartate to Casamino acids medium. It corresponds to about 2.5 seconds of replication at 37 °C, which in turn corresponds to the maximum rate of travel of the replication fork in glucose or Casamino acids media. It is proposed that only one Okazaki segment of the chromosome can be synthesized per replication fork at any instant, and that the rate of completion of such pieces sets an upper limit on the rate of chromosome replication. Replication of DNA at slow growth rates is limited by (a) the rate at which replication of the chromosome is initiated and (b) the rate at which the replication fork can initiate synthesis of 1-μ Okazaki pieces.

Water structure and the denaturation of DNA.

Nuclear magnetic resonance measurements of the water hydroxyl proton absorption area, line width and chemical shift were made to investigate the binding of water to the DNA molecule. These measurements covered a temperature range of 10 to 90°C, which included the denaturation temperature of DNA in all cases. The results indicate that the amount of water bound to DNA is very small, and is not measureably changed during denaturation. Studies of the correlation of the denaturation temperature of DNA with the structure of water and D 2 O were measured by the chemical shift of the hydroxyl proton resonance. If the chemical shift in sodium perchlorate solutions is divided into a contribution from the Na + and a contribution from the ClO 4 − , then the denaturation temperature of DNA is exactly correlated with the chemical shift due to the ion ClO 4 − . Both Na + and ClO 4 − shift the proton resonance in water to higher fields; however, the Na + has an ordering effect on the water, through hydration, while the ClO 4 − is shown to disorder water in a manner similar to an increase of temperature. The difference in the effect of Na + and ClO 4 − makes clear the difficulty in using nuclear magnetic resonance as a tool in studying changes of water structure in biological systems. The same measurement may reflect two totally different structural changes, one of biological significance, the other not. The denaturation of DNA is discussed in terms of the structure of the surrounding water. It is postulated that intra-water hydrogen bonding supports the secondary structure of DNA, thereby preventing base pairs from separating when intra-strand hydrogen bonds are broken. The probability of these bonds reforming is thus increased.

Regulation of nucleic acid synthesis in Lactobacillus acidophillus R-26

Abstract Lactobacillus acidophillus R-26 has been used to study the regulation of chromosome replication. RNA synthesis in this organism is stringent in that when amino acids are removed, RNA synthesis is drastically reduced. Synthesis can be restored by the addition of chloramphenicol. Actinomycin D inhibits RNA synthesis completely in concentrations above 2 μg/ml. DNA synthesis proceeds in the absence of all amino acids resulting in an overall increase of 40–50 % in the DNA content per cell. This synthesis can be further increased by the addition of either glutamic or aspartic acids. Thymine or deoxyribose starvation results in a 150 % increase in DNA content upon subsequent restoration of these compounds and incubation in the absence of amino acids. This increase can be inhibited by the presence of actinomycin D or chloramphenicol during the period of deoxyriboside starvation. These results are similar to those observed previously for Escherichia coli and have been explained by assuming that: (a) chromosome replication is completed in the absence of all amino acids; (b) thymine or deoxyriboside starvation induces an extra cycle of chromosome replication. It has been found that actinomycin D will inhibit the extra cycle of DNA replication, if present at concentration of 5 μg/ml. This concentration is much higher than that necessary to inhibit RNA synthesis, but much lower than that necessary to inhibit normal DNA synthesis. It is proposed that actinomycin is, in this case, acting directly upon the initiation of DNA replication.