Days weaving the lagging strand synthesis of DNA - A personal recollection of the discovery of Okazaki fragments and studies on discontinuous replication mechanism

Abstract

At DNA replication forks, the overall growth of the antiparallel two daughter DNA chains appears to occur 5'-to-3' direction in the leading-strand and 3'-to-5' direction in the lagging-strand using enzyme system only able to elongate 5'-to-3' direction, and I describe in this review how we have analyzed and proved the lagging strand multistep synthesis reactions, called Discontinuous Replication Mechanism, which involve short RNA primer synthesis, primer-dependent short DNA chains (Okazaki fragments) synthesis, primer removal from the Okazaki fragments and gap filling between Okazaki fragments by RNase H and DNA polymerase I, and long lagging strand formation by joining between Okazaki fragments with DNA ligase.

Keywords: DNA ligase; Okazaki fragments; function of RNase H and DNA polymerase 1; lagging strand synthesis; primer RNA dependent synthesis of Okazaki fragments; processing of Okazaki fragments before ligation.

Figure 1.

Models of elongation reaction of daughter DNA chains. (Case 1) One daughter chain is synthesized in the 5′-to-3′ direction while the other chain is synthesized in the 3′-to-5′ direction. Both daughter chains are synthesized continuously. This model requires a new enzyme that catalyzes the 3′-to-5′ DNA polymerization. (Case 2) Both daughter chains are synthesized in the 5′-to-3′ direction in the microscopic analytical level. The leading strand (which appears to elongate in the 5′-to-3′ direction when observed by a macroscopic analytical method) is synthesized continuously whereas the lagging strand (which appears to elongate in the 3′-to-5′ direction) is assembled from the discontinuously synthesized small DNA fragments.

Figure 2.

Strategy to determine the direction of DNA synthesis by exonuclease digestion analysis. T4 phage-infected bacteria are cultured at 20 ℃ until the peak of DNA synthesis. Bacteria are then cooled to 8 ℃ and pulse-labeled with [³H]-thymidine for 6 seconds to radiolabel the growing end of DNA and with [¹⁴C]-thymidine for 2.5 minutes to radiolabel the entire length of Okazaki fragments. The 9S short-chain DNA fraction enriched with Okazaki fragments is isolated, and the Watson-strand and Crick-strand are separated further. Each strand is subjected to digestion with E. coli exonuclease I (5′-to-3′ digestion) and Bacillus subtilis exonuclease (5′-to-3′ digestion), and the ³H and ¹⁴C radioactivity released from the DNA is counted at varying periods of digestion time. Data points are plotted for the percent release of ³H and ¹⁴C. The profiles of the ³H release indicate that the growing point locates at the 3′-end of DNA. Real results obtained are shown in Fig. 6.

Figure 3.

Okazaki fragments in E. coli. E. coli was pulse-labeled with [³H]-thymidine at 20 ℃. (a) Pulse-labeling for 10 seconds. (b) Pulse-labeling for 10 seconds followed by 2 minutes chase (i.e., an excess amount of non-radioactive thymidine was added to the culture medium). (c) Pulse-labeling for 10 seconds followed by 20 minutes chase. Cellular DNA was then denatured and extracted, and the DNA fragments were separated by length using 5–20% sucrose-gradient centrifuge in an alkaline condition. The ³H peak observed in the fractions 5–7 was the Okazaki fragments.

Figure 4.

Okazaki fragments observed in E. coli infected with DNA ligase temperature-sensitive mutant strain of T4 bacteriophage. (a) Pulse-labeling experiment at a low temperature. E. coli was infected with a gene 30 (encoding DNA ligase) temperature-sensitive T4 phage strain at 20 ℃ for 70 minutes. Bacteria were then transferred to 30 ℃ and incubated for 1 minute followed by [³H]-thymidine pulse-labeling for varying periods of time. 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. (b) Pulse-labeling experiment at a high temperature. T4 phage-infected bacteria were prepared as the panel (a) experiment and then pulse-labeled at 43 ℃. The DNA sedimentation pattern was analyzed by the alkaline sucrose-gradient centrifugation. (c) Pulse-labeling at a high temperature followed by low-temperature incubation. E. coli cells were infected with the gene 30 temperature-sensitive T4 phage mutant strain and incubated at 20 ℃ for 70 minutes. Bacteria were then transferred to 43 ℃ and incubated for 2 minutes followed by [³H]-thymidine pulse-labeling for one minute. After the labeling, cells were transferred to 30 ℃ and incubated for varying periods of time. The DNA was then extracted and analyzed by the alkaline sucrose-gradient centrifugation.

Figure 5.

Reiji Okazaki presenting the discontinuous replication mechanism at the Cold Spring Harbor Symposium in June 1968.

Figure 6.

Determination of the direction of T4 phage Okazaki fragment synthesis by exonuclease digestion analysis. T4 phage-infected bacteria were cultured at 20 ℃ until the peak of DNA synthesis. Bacteria were then cooled to 8 ℃ and pulse-labeled with [³H]-thymidine for 6 seconds (to radiolabel the growing end of DNA) and with [¹⁴C]-thymidine for 2.5 minutes (to radiolabel the entire length of Okazaki fragments). 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. coli exonuclease I (5′-to-3′ digestion) and Bacillus subtilis exonuclease (5′-to-3′ digestion), and the ³H and ¹⁴C radioactivities released from the DNA was counted at varying periods of time of digestion. Data points are plotted for the percent release of ³H and ¹⁴C. The profiles of the ³H release indicate that the growing point locates at the 3′-end of DNA.

Figure 7.

Short fragments are accumulated in the DNA ligase mutant as well as DNA polymerase mutants of E. coli in non-permissive condition. E. coli cells were cultured at 30 ℃ and then the temperature was shifted to 43 ℃, added to [³H]-thymidine for the indicated periods of time to radiolabel the newly synthesized DNA. After the indicated time, DNA was extracted from the cells and in a denaturing condition separated by length using the 5–20% alkaline sucrose-gradient centrifugation. Okazaki fragments were observed as short DNA fragments with about 10S sedimentation coefficient. Results with wild type strain; the ligts7 strain harboring a temperature sensitive DNA ligase activity; the polA12 strain harboring a temperature-sensitive polymerase activity of DNA polymerase I; the polAex1 strain harboring a temperature-sensitive 5′-to-3′ exonuclease activity of DNA polymerase I, were shown.

Figure 8.

Okazaki fragments accumulation in the pol A and rnh mutant E. coli strains. Wild type, rnh mutant, rnh and polA 5′ to 3′ exonuclease mutant of E. coli strains were grown at 30 ℃ and then transferred to 43 ℃. At this non-permissive temperature, cells were incubated in the presence of [³H]-thymidine for the indicated periods of time to radiolabel the newly synthesized DNA. After the incubation, DNA was extracted in a denaturing condition and separated by length by the 5–20% alkaline sucrose-gradient centrifugation. Okazaki fragments were observed as short DNA fragments with about 10S sedimentation coefficient. Upper left, wild type strain: upper right, the RNase H strain harboring temperature sensitive RNase H activity. Lower left, the double mutant strain harboring a temperature-sensitive 5′ to 3′ exonuclease activity of DNA polymerase 1 and RNase H activities; lower right, the polA4113 strain, which harbored a temperature-sensitive DNA polymerase I whose 5′-to-3′ exonuclease activity was suppressed under high temperature.

Figure 9.

Method of isolation of primer RNA from Okazaki fragments and determination of chain length.

Figure 10.

Detection of intact Primer RNA.

Figure 11.

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.