Hotchikiss of Rockefeller University. Okazaki fragments observed in E. DNA was then extracted from the bacterial cells, and the length of the DNA fragments were analyzed by sucrose-gradient centrifugation in the alkaline condition. The DNA sedimentation pattern was analyzed by the alkaline sucrose-gradient centrifugation. The DNA was then extracted and analyzed by the alkaline sucrose-gradient centrifugation. In , Reiji was invited to the Cold Spring Harbor Symposium, where he presented the discontinuous replication model Fig.
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 , we restarted experiments to determine the direction of the DNA synthesis at the microscopic level — the experiments that we had initially planned.
It was the era when political conflicts between angry college students and Government escalated quite violently. We labeled the full-length Okazaki fragments with [ 14 C]-thymidine, and a very short region at the growing end of the fragment was labeled with [ 3 H]-thymidine.
Determination of the direction of T4 phage Okazaki fragment synthesis by exonuclease digestion analysis. The 9S short-chain DNA fraction enriched with Okazaki fragments was isolated, and the Watson-strand and Crick-strand were separated further. Each strand was subjected to digestion with E. 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 de novo 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 E. In , Cairns et al. Nonetheless, the polA1 strain was viable under a standard culture condition.
Consequently, this mutant DNA polymerase I could not catalyze the nick translation reaction and was unable to fill the gaps in the DNA strand. Therefore, the observed accumulation of Okazaki fragments in the polA1 strain was explained by the inability of the mutant DNA polymerase I to fill the gaps between Okazaki fragments synthesized in the lagging strand. In the presence of such gaps, DNA ligases could not link Okazaki fragments to form a continuous daughter strand, resulting in the observed accumulation of Okazaki fragments in the polA1 strain.
The nick translation activity of DNA polymerase I simultaneously processed the degradation of RNA primers and the gap-filling between Okazaki fragments.
Soon, another temperature-sensitive E. It was demonstrated that this strain also accumulated Okazaki fragments when cultured at a non-permissive temperature Fig. Okazaki fragments were observed as short DNA fragments with about 10S sedimentation coefficient. Okazaki fragments accumulation in the pol A and rnh mutant E. At this non-permissive temperature, cells were incubated in the presence of [ 3 H]-thymidine for the indicated periods of time to radiolabel the newly synthesized DNA.
The greatest mystery of discontinuous replication was the mechanism of initiation of Okazaki fragment synthesis. In the s, 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. Widely accepted among the investigators specialized in the in vitro biochemical reactions was the following idea.
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.
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 was already known that all RNA polymerases can initiate polynucleotide chain synthesis without requiring a primer. 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.
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 E. 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.
While we were struggling to overcome this obstacle, another grave situation emerged. The claws of chronic myeroblastic leukemia seized upon Reiji.
He was in Hiroshima City when the Atomic Bomb was ruined the city. His reaction when he knew about his physical condition was that he was lucky to have lived 30 more years since the bombing.
Thus, we were forced to compete with the calendar — can we prove the existence of the RNA primer of Okazaki fragments before the date he has to leave? This observation convinced us of the existence of the primer RNA. In March, , Reiji and I attended a scientific meeting on DNA replication in Montebello, Canada, but by this time his leukemia turned to acute condition and was already desperate. After we had returned to Japan, Reiji was hospitalized, and on August 1st he passed away at the age of 44 without knowing the nature of the RNA primer.
Not enough time was given to him. Immediately after I had lost Reiji, I received news about a serious challenge to the existence of Okazaki fragments. Pseudo -Okazaki fragments: these are short DNA chains newly synthesized during one of the DNA repair reactions known as uracil excision repair.
The uracil base present in the DNA chain is recognized by an enzyme known as uracil-DNA glycosilase , which cuts the bond between the uracil base and sugar in the DNA backbone. The site lacking the base known as the AP site is then recognized by an endonuclease known as AP endonuclease , which cuts the backbone phosphodiester bond to induce a nick in the DNA. This final reaction of the uracil excision repair involves synthesis of a new, short DNA fragment, which will be incorporated into the continuous DNA chain by DNA ligase.
The news was that an E. This error was immediately recognized by uracil-DNA glycosylase, and the uracil excision repair reaction was initiated. Pulse-labeling experiments with 3 H-thymidine demonstrated that the sof strain frequently produced radioactive short DNA fragments, which resembled Okazaki fragments in size and hence were later called pseudo-Okazaki fragments , from the AP site.
Based on these observations, it was proposed erroneously that Okazaki fragments could actually be short DNA fragments produced by repair reaction in newly replicated region — but not the replication units in lagging-strands. It was reported by the media that, even though there was no other proper explanations available for the lagging-strand synthesis, the discontinuous replication model itself still lacks evidence for de novo synthesis of Okazaki fragments!
Secondly, the ratio of pseudo-Okazaki fragments to Okazaki fragments in the wild type E. 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 3 H-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 in vitro 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 in vivo are likely to be produced in the process of DNA repair reaction.
The discontinuous replication mechanism would not be established unless the nature of the primer was unveiled. We do not observe distributions of Okazaki fragment termini consistent with completely distinct substrates for Rad27 and Exo1. Instead, removal of Exo1 alone has a small but widespread impact on the location of Okazaki fragment termini, while the absence of Rad27 alone shifts a substantial fraction of Okazaki fragment termini towards the replication-fork proximal edge of the nucleosome Figure 2.
Importantly, the most common location of termini in the absence of both nucleases is distinct from that observed in the sole absence of either nuclease Figure 2. Thus, fragments whose processing is impaired by the absence of Rad27 can apparently still be at least partially processed by Exo1. We propose that distinct Okazaki nucleases do not have rigid substrate preferences, but instead compete to cleave lagging-strand substrates in vivo. The absence of Rad27 reduces strand displacement more than the absence of Exo1.
Consistent with this model, temperature-sensitive dna2 strains are not mutators 17 and depletion of Dna2 does not significantly alter global strand displacement in vivo Figures 2 and 3. We observe that the contribution of Dna2 to Okazaki fragment processing is extremely limited in the uniquely mappable regions of the genome that can be assayed using our sequencing methodology Figures 2 and 3. Therefore, although the nuclease activity of Dna2 is essential for viability in checkpoint-proficient cells 34 , the absence of Dna2 during S-phase is not obviously detrimental to exponentially dividing S.
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