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.
It took several interesting experiments by Frederick Griffith, Avery, MacLeod, McCarty, Alfred Hershey, Martha Chase etc., to discover that DNA is the hereditary material.
discontinuous replication, Okazaki fragments, DNA ...
We have analyzed the transition sites between primer RNA and DNA in a 589 bp segment of the bacteriophage T7 genome. In the monomeric replication stage, RNA-DNA transition sites are predominantly on the light (L) strand (with, 5′→3′ polarity on the genetic map) but rarely on the heavy (H) strand, indicating that replication proceeds semidiscontinuously with the H and L strands corresponding to the leading and lagging strands, respectively. The direction of replication is that expected from the position of the primary origin and also indicates that secondary origins are seldom if ever used. In the concatemeric stage of replication, RNA-DNA transition sites are instead distributed on both strands of the segment with equally high frequency, showing that initiation occurs within the concatemeric molecule per se and by a different mechanism.
DNA Replication: The Leading Strand and DNA …
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.
A summary of DNA Replication in 's DNA Replication and ..
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.
DNA replication of one helix of DNA results in two ..
In 1968, Reiji was invited to the Cold Spring Harbor Symposium, where he presented the discontinuous replication model (). At that time, the DNA synthesis reaction at the replication fork was considered a major biological mystery. The chair of the symposium even included in his keynote address a slide showing a picture of the fork partly hidden by a fig leaf. Our discontinuous replication model was accepted as a major clue to the solution of this problem and became one of the highlights of the symposium. In this meeting, the term was given to the short DNA fragments that appear during the lagging strand synthesis, and this name has remained generally accepted even in today’s textbooks.