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.
Dr. Lark, Kansas State University, attended the International Congress of Biochemistry. He offered us a research opportunity in his laboratory, so we stayed in the U.S. for about six months in the autumn of 1967 as a visiting professor and a visiting associate professor of Kansas State. On the way to Kansas, we stopped by the Kornberg laboratory and were notified of the discovery of DNA ligase, the enzyme that forms a covalent phosphodiester bond between the 3′- and 5′-termini of DNA chains. This enzyme had characteristics that were expected of an enzyme that forms links between Okazaki fragments. Dr. Richardson of Harvard University, who had been our friend since our stay in Stanford, discovered that gene of bacteriophage T4 (a virus that infects bacterial cells) encoded DNA ligase, and he kindly provided us with a temperature-sensitive mutant T4 phage strain whose gene product became dysfunctional at high temperatures. We started experiments using this mutant phage strain immediately after we had arrived Kansas.
In E. coli, there are two pathways by which the DnaB helicase is loaded onto the lagging-strand template DNA: One is primosome formation directed by the PriA protein, and the other is DnaA protein-directed DnaB loading (8, 9). The former process was discovered first in the replication of bacteriophage fX174 DNA and plasmid ColE1 DNA, and it later appeared to be involved in the resumption of chromosomal DNA replication after replication of the E. coli genome has been interrupted or halted. On the other hand, the latter process was found in oriC plasmid DNA replication in vitro and is thought to form a priming complex with DnaG primase at the replication fork in E. coli chromosomal DNA replication.
There is another difference in the enzymatic processes of synthesizing the leading and lagging strands. Leading-strand DNA synthesis requires RNA primer only once in the replication of each replicon, but a frequent priming process is associated with lagging-strand DNA synthesis. RNA primer must be laid down as the initiation step of each cycle of synthesis of Okazaki fragments. Therefore, the priming proteins, including the primase, are required for lagging-strand DNA synthesis, along with the replication fork.
The chain elongation of Okazaki fragments in E. coli is catalyzed by DNA polymerase III holoenzyme (10). This enzyme possesses a capacity to synthesize DNA with a very high processivity, sufficient for completion of about 2 kb of Okazaki fragment. In addition, the Pol III holoenzyme dissociates from the nascent Okazaki fragment and restarts the next round of Okazaki fragment synthesis from an RNA primer newly settled near the replication fork (11). Enzymes to remove primer RNA and fill the gap, such as ribonuclease H and DNA polymerase I of E. coli, are essential for the sealing of Okazaki fragments by DNA ligase (12). Mutants defective in either DNA polymerase I or DNA ligase show a massive accumulation of short Okazaki fragments under restrictive conditions.
We assumed that the primer RNA segment may attach to the 5′-end of Okazaki fragments and attempted to detect this RNA. However, soon we encountered a great difficulty. The number of Okazaki fragments associated with the primer RNA segment turned out to be extremely low (only about 10 molecules in a wild type cell), and the length of the primer RNA was far shorter than it had initially been predicted (less than 10 nucleotides). Moreover, enormous numbers of RNA fragments with various lengths, which we called free RNA, were found in the bacterial cells. Only a trace amount of contamination of such intracellular free RNA would result in a serious experimental artifact.
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.
Important insights came from reports that initiation of DNA synthesis in retroviruses and M13 bacteriophage involved RNA., These discoveries prompted us to presume that the events initiating Okazaki fragment synthesis may also involve RNA. It was already known that all RNA polymerases can initiate polynucleotide chain synthesis without requiring a primer. Moreover, DNA polymerase can utilize an RNA polynucleotide chain as a primer, as long as the RNA forms a heteroduplex structure with the complementary DNA chain. However, rifampicin, an inhibitor of bacterial RNA polymerase that specifically binds to the β-subunit of the enzyme, did not inhibit DNA chain elongation occurring in the chromosome. Therefore, our hypothesis required a new RNA polymerase that was resistant to rifampicin. As a matter of fact, the primase enzyme, which synthesizes very short RNA primer chains on the single-stranded DNA template, was discovered later, and it was indeed a new type of RNA polymerase that was resistant to rifampicin.
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.
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.