explain why continuous synthesis of both DNA strands is not possible
25/02/2009 · Why is continuous synthesis of both DNA strands not possible
why continuous synthesis of both DNA strands is not possible.
The discontinuous replication model was originally proposed to explain the mechanism of the lagging strand synthesis. However, based on the observations that DNA chains synthesized in bacteria deficient of DNA ligase or DNA polymerase I were all short, the possibility of the both-strand discontinuous replication was once considered. However, because both of DNA ligase and DNA polymerase I are involved in the DNA repair process, it was later interpreted that the incorporation of the 3H-labeled thymidylate into exclusively into short DNA fragments in the absence of these enzymes would not necessarily support the double-strand discontinuous replication. Extrapolating from the products in the reaction with purified replication enzymes, majority of investigators now believe that the leading strand is synthesized in the continuous manner only, and that the leading strand-derived radioactive short DNA fragments generated are likely to be produced in the process of DNA repair reaction.
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For example, synthesis of Ag nanoparticles at a reaction temperature of 25 °C via (sweet orange) peel extract produced particles with an average size of around 35 nm.
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Ms. Sakabe, Reiji’s first graduate student, performed the low-temperature pulse-labeling experiment using . Unexpectedly, the [3H]-thymidine radioactivity was incorporated into short DNA fragments that were only 1,000–2,000 nucleotides in length (). These newly synthesized short DNA fragments are now known as Okazaki fragments. When the pulse-labeling time was extended or the radiolabeling was chased by non-radioactive thymidine, the tritium radioactivity was transferred from the short DNA fragments to longer DNA chains that showed physical characteristics identical to the overall genomic DNA. These results suggested that the short DNA fragments were synthesized at the very early stage of DNA replication reaction and, only after completion of their synthesis, these DNA fragments were incorporated into the long and continuous chains of genomic DNA — , the . We obtained these results in 1966, after three years of efforts. When we presented these data in a domestic meeting, we received a comment that the observed short DNA fragments could be artifacts derived from the fragile DNA strands near the replication fork. To address this, we repeated the pulse-labeling experiments using a variety of systems and tested various protocols of cell lysis and DNA extraction. Still, all results suggested the existence of the short DNA fragments. Next year (1967), at the International Congress of Biochemistry in Tokyo, we presented the discontinuous model of DNA replication.
5e) Twice for a cell with two pairs of chromosomes: once showing that thechromosome with goes with the chromosome carrying (and with ), and a second time showing going with and with . Note that the outcome depends on the alignment of chromosomes inthe first metaphase plate, and that this alignment occurs at random. [Note thatthere are so many genes in each chromosome that, in practice, no two members ofa pair are identical; i.e. there will be many genes for which the chromosomeswill have different alleles); note also that the number of possible outcomesgoes up exponentially with the number of pairs of chromosomes; thus, for 2n = 6the number of possibilities is 4 (22); for 2n = 46 (as in humans) thetotal number is 222.]
Repair synthesis by DNA pol fills in the missing nucleotides.
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At the Salk Institute for Biological Studies, in 1994, Leslie Orgel observes, "Because synthesizing nucleotides and achieving replication of RNA under plausible prebiotic conditions have proved so challenging, chemists are increasingly considering the possibility that RNA was not the first self replicating molecule..." .
We stayed in Stanford only for 15 months (December 1961–Early March 1963). Although this was a great opportunity to learn details of the biochemical reactions and technologies of DNA polymerase, we simultaneously learned that DNA replication could not be explained solely by the reactions of the DNA polymerase. DNA polymerase cannot unwind the DNA double helix, and the intact double-stranded DNA, which must be the genuine replication template , does not serve as template for the DNA polymerase reaction . With the double-stranded DNA template, synthesis of new DNA is observed only at the nicks or gaps of the template. Besides, DNA polymerase cannot synthesis of a new DNA chain — namely, this enzyme requires a polynucleotide and is only able to the primer chain — and the DNA synthesis occurs only at the 3′-OH termini. Therefore, DNA synthesis by DNA polymerase occurs only in the 5′-to-3′ direction (, tail growth); the 3′-to-5′ chain elongation, or head growth, is never observed. Prolonged DNA polymerase reaction produces branched DNA because of template-switching, a phenomenon in which DNA polymerase suddenly switches its template strand from one antiparallel DNA chain to the other. The products of such replication reactions are abnormal DNA molecules that cannot be denatured.
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Prompted by the discovery of the discontinuous replication, the biochemical research on DNA replication after the 1970s was led by efforts to reconstitute the reactions at the replication fork . The major achievements of this era include elucidation of the mechanism of the primer synthesis, reconstitution of the processive DNA-synthesizing machinery that mimics the velocity of replication (1,000 nucleotides per second), and reconstitution of the replication fork protein complex that synthesizes both the leading and lagging strands simultaneously. The precise device of the fork reactions and the common mechanism of DNA replication conserved among the prokaryotes and eukaryotes are examples of research themes that have long attracted investigators.–
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The steps of the discontinuous replication mechanism elucidated by the above research are shown in . Our research, from the discovery of Okazaki fragments to the establishment of the series of steps in the replication reaction, was performed by employing the approach of analyzing the DNA products of the reactions occurring in cells.
What Is DNA Replication? - Conservative, Semi …
Widely accepted among the investigators specialized in the biochemical reactions was the following idea. As described earlier in this essay, prolonged DNA replication reaction catalyzed by DNA polymerase I produces branched-form DNA because of the template-switching phenomenon. They assumed that the same template-switching was taking place at the replication fork. That is, a DNA polymerase enzyme that has been synthesizing the leading-strand daughter chain in a continuous fashion switches the template strand spontaneously at a certain frequency. As a consequence of the template switching, the same DNA polymerase I is now synthesizing the lagging strand by simply adding nucleotides, still in a continuous fashion, to the end of the same DNA strand that it was synthesizing moments before as the leading strand. This forms a hairpin-like structure of the single-stranded daughter DNA, of which 5′-half is the leading strand and the 3′-half is the lagging strand, at the replication fork. The hairpin-shaped, single-stranded daughter DNA will then be cut at the junction between the leading and lagging strands, thus leaving an Okazaki fragment as a precursor of the lagging strand, and the DNA polymerase I goes back to the task of synthesizing the leading strand, again by the spontaneous template switching. By repeating the above processes, both the leading and lagging strands of daughter DNA appear to be synthesized simultaneously. Importantly, this hypothetical model (which is considered incorrect today) did not require frequent initiation of DNA synthesis, and it even explained the origin of Okazaki fragments.
How do we know that DNA replication is semi-conservative
Steps of the discontinuous DNA replication reaction. The leading strand is synthesized continuously while the lagging strand is synthesized discontinuously. The elongation reaction of the lagging strand consists of five steps: I, Unwinding of the DNA template; II, Primer synthesis; III, DNA (Okazaki fragment) synthesis; IV, Primer degradation and gap filling; and V, Ligation of Okazaki fragments. The dots on the template DNA indicate the signal sequences for primer RNA synthesis.
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