Continuous DNA synthesis

In mammalian cells, at least eight DNA polymerases are present. DNA polymerase a is involved in the initiation of DNA synthesis at DNA replication origins and lagging strand synthesis (Wang, 1991). DNA polymerase 7is a mitochondrial DNA polymerase (Wang, 1991). Recently, bypass polymerases, such as DNA polymerase V, t. and Chave also been identified (Lindahl and Wood, 1999). These DNA polymerases are capable of continuing DNA synthesis even through bulky DNA lesions—such as UV-induced pyrimidine-dimers in the template strand (Lindahl and Wood, 1999). 

Tetrahydrofolate cofactors participate in one-carbon transfer reactions. As described above in the section on vitamin B12, one of these essential reactions produces the dTMP needed for DNA synthesis. In this reaction, the enzyme thymidylate synthase catalyzes the transfer of the one-carbon unit of N 5,N 10-methylenetetrahydrofolate to deoxyuridine monophosphate (dUMP) to form dTMP (Figure 33-2, reaction 2). Unlike all of the other enzymatic reactions that utilize folate cofactors, in this reaction the cofactor is oxidized to dihydrofolate, and for each mole of dTMP produced, one mole of tetrahydrofolate is consumed. In rapidly proliferating tissues, considerable amounts of tetrahydrofolate can be consumed in this reaction, and continued DNA synthesis requires continued regeneration of tetrahydrofolate by reduction of dihydrofolate, catalyzed by the enzyme dihydrofolate reductase. The tetrahydrofolate thus produced can then reform the cofactor N 5,N 10-methylenetetrahydrofolate by the action of serine transhydroxy- methylase and thus allow for the continued synthesis of dTMP. The combined catalytic activities of dTMP synthase, dihydrofolate reductase, and serine transhydroxymethylase are often referred to as the dTMP synthesis cycle. Enzymes in the dTMP cycle are the targets of two anticancer drugs methotrexate inhibits dihydrofolate reductase, and a metabolite of 5-fluorouracil inhibits thymidylate synthase (see Chapter 55 Cancer Chemotherapy). 

In bacteria it has been demonstrated that protein synthesis is not essential for the continuation of a cycle of DNA synthesis already in progress (Hanawalt et al., 1961 Lark, 1963). In contrast, continued DNA synthesis in eukaryotes appears to be closely dependent on continued protein synthesis. In Physarum polycephalum, where the S phase... 

It is unlikely that protein synthesis is required solely for the initiation of DNA synthesis at specific chromosomal sites during the S phase in order that replication continue. The inhibition of cellular protein synthesis would affect many metabolic functions, causing gradual cessation of cell activity. Closely associated with DNA replication is the synthesis of chromosomal proteins necessary for the formation of new chromosomes. Different histone fractions, for example, show differences in their degree of dependence on DNA synthesis (Sagopal and Bonner, 1969). The converse may also hold continued DNA synthesis may be dependent on the availability of specific proteins, possibly for the incorporation of newly synthesized DNA into new chromosomal structure. 

When heterokaryons are formed between animal cells the nuclei in the same cytoplasm undergo synchronous initiation of DNA synthesis (Harris and Watkins, 1965) similar synchrony is observed in binucleate cells occurring in mouse embryo cultures (Church, 1967). DNA synthesis has been examined in multinucleate HeLa cells formed by fusion between cells in different phases of the life cycle (Rao and Johnson, 1970). There was a rapid induction of DNA synthesis in G1 phase nuclei following the fusion of G1 and S phase cells. When S phase cells were fused with G2 phase cells, no induction of DNA synthesis was observed in G2 phase nuclei and, no matter what the ratio of G2 S nuclei in the fused cell, the S phase nuclei continued DNA synthesis. 

DNA synthesis occurs in both directions at each of the rep-licating forks. Once a DNA strand has been primed, synthesis toward the replicating fork can be visualized as continuous. Growth of the opposite,... 

Figure 4.26 (a) DNA replication at low resolution (for example as seen by electron microscopy). Only one replication fork is visible and it appears that both strands of the parental DNA replicate continuously in the same direction, which cannot be the case, since the two strands of parental DNA are anti-parallel, (b) The problem is solved by the priming of DNA synthesis with short RNA primers, whose 3 -hydroxyl can be used by DNA polymerase, producing Okazaki fragments, while on the other strand, DNA synthesis is continuous. (From Voet and Voet, 2004. Reproduced with permission from John Wiley Sons., Inc.)... 

In higher eukaryotes, at the onset of S phase cyclin A accumulates which stimulates DNA synthesis. The amount of cyclin A continues to be high after the S phase because of its role in chromosome condensation. Cyclin A is degraded when cells enter prometaphase. The level of another cyclin called cyclin B rises during G2 phase, which helps to complete the chromosome condensation and spindle assembly, which allow transition to metaphase. Cyclin B is degraded by APC during metaphase. ... 

This section examines the synthesis of nucleosides that contain seven-membered sugar analogues in place of the deoxyribose component. Nucleosides from the last group have been further incorporated into ONs via solid-phase DNA synthesis. A physical and biochemical investigation of the oligomers thus prepared continues in the next section. The study under review culminated in the assessment of the ability of the oligomers to complex with single-stranded RNA and for the heteroduplexes so formed to serve as substrates of RNAseH. 

The classic example of schedule dependency is cy-tarabine, a drug that specifically inhibits DNA synthesis and is cytotoxic only to cells in S-phase. Continuous infusion or frequent administration of cytarabine hydrochloride is superior to intermittent injection of the drug. Bleomycin is another drug for which continuous infusion may increase therapeutic efficacy. 

Male and female Fischer 344 rats and B6C3Fi mice were fed di(2-ethylhexyl) phthalate for up to 13 weeks (David et al., 1999). In rats fed 12 500 ppm di(2-ethylhexyl) phthalate, there was an increase in hepatocyte replicative DNA s mthesis (measured after continuous bromodeoxyuridine administration (osmotic pump) for three days before sampling) after one week (but not after two or 13 weeks) and an increase in hepatic peroxisomal [3-oxidation activity after one, two and 13 weeks administration. In mice fed 10 000 and 17 500 ppm di(2-ethylhexyl) phthalate, there was no increase in hepatocyte replicative DNA synthesis (measured after continuous bromodeoxyuridine three days before sampling) after one, two or 13 weeks of administration, but there was an increase in hepatic peroxisomal 3-oxidation activity after one, two and 13 weeks administration. In mice fed 1000 ppm di(2-ethylhexyl) phthalate, there was no statistically significant increase in hepatic peroxisomal 13-oxidation activity after one, two or 13 weeks administration (bromodeoxyuridine labelling was not evaluated at this lower dietary concentration of di(2-ethylhexyl) phthalate). 

In view of the short plasma half-life of 5-FU, most authors believe that continuous administration of 5-FU (CIFU) is the superior 5-FU schedule compared to bolus regimens. Continuous infusion of 5-FU seems to inhibit predominantly DNA s mthesis, whereas bolus administration of 5-FU inhibits RNA splicing and DNA synthesis, resulting in different toxicity and efficacy profiles (24,25). 

FIGURE 26-7 Model for p-independent termination of transcription in f. coli. RNA polymerase pauses at a variety of DNA sequences, some of which are terminators. One of two outcomes is then possible the polymerase bypasses the site and continues on its way, or the complex undergoes a conformational change (isomerization). In the latter case, intramolecular pairing of complementary sequences in the newly formed RNA transcript may form a hairpin that disrupts the RNA-DNA hybrid and/or the interactions between the RNA and the polymerase, resulting in isomerization. An A=U hybrid region at the 3 end of the new transcript is relatively unstable, and the RNA dissociates completely, leading to termination and dissociation of the RNA molecule. This is the usual outcome at terminators. At other pause sites, the complex may escape after the isomerization step to continue RNA synthesis. 

For the very low density varieties of the cases shown in Figure 2 and, more particularly, Figure 4 (curve 7), for which initiation is slow compared to both termination (release from end of template) and polymerization, a simpler treatment, in which the interference of one ribosome with another is totally neglected, should suffice. In this case an equation of the form of Eq. (1), herein only applied to the problem of DNA synthesis, should be valid, but Eqs. (2) and (3) should be modified to account for repetitive initiation at site 1 and continuing release from site K, respectively Eqs. (4) and (6) will not apply. In the even more restricted (but perhaps biochemically relevant) case in which, in addition to neglecting ribosome interference, one may also neglect the back reaction (kb x 0), one may solve this system of equations (Eq. (1), plus Eqs. (2) and (3) modified as described) very easily by taking Laplace transforms.13 This is the only case with repetitive initiation for which we have been able to find solutions for the transient, as well as steady state, behavior. 

Many bacterial cells contain self-replicating, extrachromosomal DNA molecules called plasmids. This form of DNA is closed circular, double-stranded, and much smaller than chromosomal DNA its molecular weight ranges from 2 X 106 to 20 X 106, which corresponds to between 3000 and 30,000 base pairs. Bacterial plasmids normally contain genetic information for the translation of proteins that confer a specialized and sometimes protective characteristic (phenotype) on the organism. Examples of these characteristics are enzyme systems necessary for the production of antibiotics, enzymes that degrade antibiotics, and enzymes for the production of toxins. Plasmids are replicated in the cell by one of two possible modes. Stringent replicated plasmids are present in only a few copies and relaxed replicated plasmids are present in many copies, sometimes up to 200. In addition, some relaxed plasmids continue to be produced even after the antibiotic chloramphenicol is used to inhibit chromosomal DNA synthesis in the host cell. Under these conditions, many copies of the plasmid DNA may be produced (up to 2000 or 3000) and may accumulate to 30 to 40°/o of the total cellular DNA. 

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