Discontinuous DNA synthesis

As mentioned earlier, discontinuous DNA synthesis necessitates the existence of an enzyme for joining the newly synthesized segments (see fig. 26.6). Such an enzyme has been found in a variety of cell types and is called polynucleotide ligase (fig. 26.9). 

Okazaki and his colleagues provided the experimental evidence for discontinuous DNA synthesis.) Subsequently, these pieces (now called Okazaki fragments) are covalently linked together by DNA ligase. (In prokaryotes such as E. coli, Okazaki fragments possess from 1000 to 2000 nucleotides.)... 

Tseng, B. Y., and Goulian, M., 1977, Initiator RNA of discontinuous DNA synthesis in human lymphocytes. Cell 12 483. 

Figure 36-16. The discontinuous poiymerization of deoxyribonucleotides on the lagging strand formation of Okazaki fragments during iagging strand DNA synthesis is illustrated. Okazaki fragments are 100-250 nt iong in eukaryotes, 1000-2000 bp in prokaryotes.

DNA synthesis proceeds in a 5 — 3 direction on each strand of the parental DNA. On the strand with 3 —>5 orientation (the leading strand) the new DNA is synthesized continuously. On the strand that has 5 —>3 orientation (the lagging strand) the DNA is synthesized discontinuously as a series of short Okazaki fragments that are then joined together. 

Unlike the DNA polymerases, RNA polymerase is able to initiate a new RNA chain, using DNA as a template (Chap. 17). The DNA polymerases are able to extend the DNA from an RNA primer. In discontinuous DNA chain growth, a particular type of RNA polymerase, called primase in E. coli, lays down short RNA primers at fairly regular base intervals, as unwinding of the helix at the replication fork proceeds. These primers are involved in the initiation of synthesis of nascent DNA chains by DNA polymerase. 

DNA replication in E. coli starts at a unique origin (oriC) and proceeds sequentially in opposite directions. More than 20 proteins are required for replication. An ATP-driven helicase unwinds the oriC region to create a replication fork. At this fork, both strands of parental DNA serve as templates for the synthesis of new DNA. A short stretch of RNA formed by primase, an RNA polymerase, primes DNA synthesis. One strand of DNA (the leading strand) is synthesized continuously, whereas the other strand (the lagging strand) is synthesized discontinuously, in the form of 1-kb fragments (Okazaki fragments). Both new strands are formed simultaneously by the concerted actions of the highly processive... 

A causal relationship between lithium treatment and hyperparathyroidism has been suggested [37]. Lithium seems to induce morphological changes in the parathyroid glands with an increase in parathyroid volume, and an increase in cellular DNA synthesis [37-39], which may explain why its effects may not be completely reversible. It is not rare to find patients with hypercalcemia, usually mild, after discontinuation of prolonged lithium therapy. A number of cases have been reported where hypercalcemia and hypocalciuria persisted even after discontinuation. We also have seen persistence of hypercalcemia and hyperparathyroidism several years after discontinuation of lithium therapy [Batlle et al unpublished, 2000]. 

What characteristic of DNA results in the requirement that some DNA synthesis is discontinuous How are Okazaki fragments and DNA ligase utilized by the cell ... 

Schematic drawing of the replication fork showing continuous 50 to 30 synthesis on the leading strand and discontinuous 50 to 30 DNA synthesis on the lagging strand through the formation of Okazaki fragments.

DNA replication is semiconservative and proceeds bidirectionally from an origin of replication. DNA synthesis is always in the 50 to 30 direction relative to the template strand. A replication fork consists of a leading strand in which DNA synthesis is continuous, and a lagging strand characterized by the discontinuous formation of short Okazaki fragments. 

Lagging strand The template strand of DNA which is copied discontinuously during DNA synthesis to produce Okazaki DNA fragments. 

Okazaki fragments Short DNA segments generated during discontinuous replication of the lagging strand these were described by Reiji Okazaki, based on experiments using radioactive deoxynucleotides as precursors in DNA synthesis. 

The direction of DNA synthesis is from 5 end to the 3 end of the newly formed strand. One strand (the leading strand) is formed continuously, while the other strand (the lagging strand) is formed discontinuously. On the lagging strand, small fragments of DNA (Okazaki fragments) are subsequendy linked. 

In conclusion, an inhibitor of thymidylate synthesis and uptake and thus of DNA synthesis has been found. It consists of two components, methotrexate and uridine. It inhibits DNA synthesis to the extent and in ways which lead to blockage of the next cell division. This would be expected if DNA replication itself or events coupled to progression in the S phase are required to take the cell on to division. Inhibition by methotrexate + uridine can be discontinued by addition of excess thymidine, or just by washing of the cells with fresh growth medium, with or without added folate. These agents shall be used as tools in attempts to control DNA replication independently of cell division in populations with temperature-synchronized cell division. 

This report has indicated that truly synchronous cell division is limited to the major fraction of cells that initiates replication prior to a time point midway between the two last heat shocks. At this time the population lacks the information that the heat treatment will be discontinued after the next heat shock, and cells continue to engage asynchronously in DNA synthesis. Experiments indicate that this occurs until around the time (EH + 40 minutes) when the next heat shock would have occurred. We have argued that cells that replicate late in the program of heat shocks perturb the division synchrony and subsequently perturb the DNA replication synchrony. It is therefore suggested that further work on temperature synchronization of Tetrahymem cell division should be directed toward the goal of preventing new engagement in DNA replication after a critical time in advance of the synchronous division. 

Theoretically, discontinuous synthesis is not necessary for the progress of replication along the 3 ->5 replicating parent strand, but there is evidence that this may also be discontinuous. In addition to normal DNA-dependent DNA synthesis, RNA-dependent synthesis can occur in some cases (see RNA-depen-dent DNA polymerase). [A.Komberg DNA Replication, W.H.Freeman Co., 1980 A.Komberg 1982 Supplement to DNA Replication, W.H.Freeman Co., 1982]... 

A second possibility to account for the observed defect in DNA synthesis during growth at low unsaturated acid levels is that the decreased fluidity of the membrane under these conditions directly affects the process of mitochondrial DNA replication. It has been suggested that mitochondrial DNA synthesis is a membrane-associated process. Recent work in our laboratory has demonstrated that the fluidity of the mitochondrial membrane has a direct influence on mitochondrial DNA replication in vitro An Arrhenius plot of DNA replication in vitro has a discontinuity (Fig. 5), the temperature of which varies with the unsaturated fatty acid content of the mitochondrial membranes (Table II). These results demonstrate the existence of a close functional relationship between mitochondrial DNA replication and the physical properties of the mitochondrial membrane. 

At each replication fork there are two DNA polymerase complexes. As the double-stranded DNA is unwound, two template strands are exposed. One of the templates can be replicated in a continuous fashion by DNA polymerase since a continuous synthesis of new strands can occur in the 5 to 3 direction as the template strand is exposed. Since all growing chains must be synthesized in file 5 to 3 direction, the lagging chain must be continuously reinitiated as new template is exposed. The lagging strand is then synthesized discontinuously, in pieces that must be joined together later. 

The lagging strand is synthesized discontinuously as a series of small fragments (about 1,000 nucleotides long) known as Okazaki fragments. Each Okazaki fragment is initiated by the synthesis of an RNA primer by primase, and then Completed by the synthesis of DNA using DNA polymerase III. Each fr ment is made in the 5 - 3 direction. 

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