Events at a DNA replication Fork -- Discontinuous synthesis ..

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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Debunking Evolution: Problems between the ..

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The greatest mystery of discontinuous replication was the mechanism of initiation of Okazaki fragment synthesis. In the 1970s, it became increasingly clear that all DNA polymerases always require primers for initiation of their polymerase reaction and that none of them can initiate DNA polynucleotide chain synthesis from only two nucleotides. As synthesis of Okazaki fragments must be initiated frequently during the process of DNA replication, we had no clues as to how to explain the biochemical basis of such events.

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When the discontinuous replication model was proposed, DNA polymerase I was the only DNA polymerase enzyme identified in . However, it was soon recognized that the DNA polymerization reaction catalyzed by this enzyme required a primer, a pre-existing short polynucleotide chain. In other words, DNA polymerase I is only capable of adding a nucleotide to the end of a pre-existing polynucleotide chain. For the true initiation of the DNA replication reaction, the existence of another DNA polymerase enzyme that is capable of the synthesis of the polynucleotide chain was anticipated. That is, DNA chain synthesis that can be initiated without requiring a pre-existing polynucleotide precursor. When the cell components were separated into the soluble or membrane fractions under mild conditions, most of the DNA polymerase I activity was recovered in the soluble fraction, but the membrane fraction still contained the discontinuous replication activity.

The continual synthesis of the ..

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.

can carry out DNA synthesis on discontinuous ..

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.

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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.