Slow Joining of Newly Replicated DNA Chains in DNA Polymerase I-Deficient
            <i>Escherichia coli</i>
            Mutants

Okazaki, Reiji; Arisawa, Mikio; Sugino, Akio

doi:10.1073/pnas.68.12.2954

Cited by 143 publications

(71 citation statements)

References 33 publications

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“…This interpretation was confirmed in polA mutants, deficient in DNA polymerase I (13)(14)(15) and with DNA ligase mutants (11,16,17). Not only was all DNA synthesized in LMW pieces in polA and ligA mutants, but it then stayed short for some time, indicating that both DNA polymerase I and DNA ligase are the maturation functions.…”

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confidence: 51%

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The Replication Intermediates in Escherichia coli Are Not the Product of DNA Processing or Uracil Excision

Amado

Kuzminov

2006

Journal of Biological Chemistry

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The current model of DNA replication in Escherichia coli postulates continuous synthesis of the leading strand, based on in vitro experiments with purified enzymes. In contrast, in vivo experiments in E. coli and its bacteriophages, in which maturation of replication intermediates was blocked, report discontinuous DNA synthesis of both the lagging and the leading strands. To address this discrepancy, we analyzed nascent DNA species from ThyA ؉ E. coli cells replicating their DNA in ligase-deficient conditions to block maturation of replication intermediates. We report here that the bulk of the newly synthesized DNA isolated from ligase-deficient cells have a length between 0.3 and 3 kb, with a minor fraction being longer that 11 kb but shorter than the chromosome. The low molecular weight of the replication intermediates is unchanged by blocking linear DNA processing with a recBCD mutation or by blocking uracil excision with an ung mutation. These results are consistent with the previously proposed discontinuous replication of the leading strand in E. coli.Before the polarity of DNA strand extension was established, both DNA strands at the replication fork were thought to be synthesized continuously, being extended by two distinct DNA polymerases with opposite polarities (1, 2) (Fig. 1A). The finding that DNA polymerases extend DNA strands only in the 5Ј to 3Ј direction (reviewed in Ref.3) made it clear that at least the DNA strand synthesized in the direction opposite to the one of the replication fork movement has to be replicated in pieces and then assembled (maturated) into a full-length molecule (4 -6). Since this strand could not be synthesized continuously, its synthesis would lag behind the apparently continuous synthesis of the opposite DNA strand, which would thus lead the replication fork progress. The two strands were eventually called the "lagging" and the "leading" strands, respectively, and the hypothetical difference in their synthesis formed the basis of the semidiscontinuous model of DNA replication (Fig. 1B) (7). However, the semidiscontinuous paradigm is based on in vitro results and ignores a large body of in vivo data, which is reviewed below.Okazaki and colleagues (1, 8) used pulses of [ 3 H]thymidine to characterize the newly synthesized DNA of wild type Escherichia coli and Bacillus subtilis strains as well as E. coli bacteriophage T4. The cells were grown at 20°C to slow down the maturation of replication intermediates into the full-length DNA molecules, and short (10 -60-s) pulses of label were used (1, 8 -11). Using alkaline (denaturing) sucrose gradients to separate replication intermediates from their template strands, Okazaki and colleagues observed that the newly synthesized DNA after 2-10 s of labeling migrated mostly as low molecular weight (LMW) 2 species, with a mean length between 1 and 2 kb (1, 8). A chase with nonradioactive thymidine shifted the label into high molecular weight (HMW) DNA, proving that the LMW species are true replication intermediates (5, 8). These experi...

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confidence: 51%

“…With this evidence, Okazaki and colleagues proposed a discontinuous model of DNA replication (Fig. 1C) in which both the lagging and the leading strands are synthesized in short fragments and maturated later into HMW species by the combined action of DNA polymerase I (plus RNase H) and DNA ligase (15,19).…”

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confidence: 99%

The Replication Intermediates in Escherichia coli Are Not the Product of DNA Processing or Uracil Excision

Amado

Kuzminov

2006

Journal of Biological Chemistry

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“…Evidence has accumulated that DNA is replicated by a discontinuous mechanism involving systhesis and joining of short DNA units (1)(2)(3)(4)(5)(6)(7)(8). Little is known, however, about the mechanism of creation of the short DNA chains.…”

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confidence: 99%

RNA-Linked Nascent DNA Fragments in Escherichia coli

Sugino

Hirose

Okazaki

1972

Proc. Natl. Acad. Sci. U.S.A.

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203

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Evidence has accumulated that DNA is replicated by a discontinuous mechanism involving systhesis and joining of short DNA units (1-8). Little is known, however, about the mechanism of creation of the short DNA chains.We postulated (2) that short DNA units might be synthesized by a mechanism in which specific structures on the template strands serve as initiation or termination signals. Although short DNA similar to that found in vivo can be produced and joined in crude in vitro systems (refs. 9-13, Ogawa and Okazaki, to be published), synthesis of such DNA has not been demonstrated with purified DNA polymerases. Unlike RNA polymerases, the known DNA polymerases do not seem to initiate new chains along parental templates, but rather catalyze the extention of a preexisting chain (14-18). Furthermore, DNA polymerases have relatively poor template specificities (18,19), in contrast to those of RNA polymerases and other proteins involved in transcription (20,21). These facts, as well as the recent findings of Kornberg and coworkers (22, 23) indicating a requirement for RNA synthesis to produce a primer for initiation of replication of phage M13 DNA, suggest that RNA synthesis (transcription) might be involved in the initiation of synthesis of the short DNA chains at specific sites on the template in the discontinuous mode of replication. In accord with this possibility, we have obtained evidence that a short stretch of RNA is covalently linked to the nascent DNA fragments. MATERIALS AND METHODSThe following Escherichia coli strains were used: AB301 (Hfr met-X+); D10

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“…These mutants are abnormally sensitive to ultraviolet irradiation and to the alkylating agent methyl methanesulfonate, and show retarded joining of the 10S "Okazaki fragments" (6)(7)(8). A recent analysis of partially purified fractions derived from..several of the polA mutants has demonstrated that although they contain much reduced levels of DNA polymerase I activity the 5' --3' exonuclease associated with the polymerase I is present in nearly normal amounts (9).…”

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confidence: 99%

A Conditional Lethal Mutant of Escherichia coli K12 Defective in the 5′ → 3′ Exonuclease Associated with DNA Polymerase I

Konrad

Lehman

1974

Proc. Natl. Acad. Sci. U.S.A.

141

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A mutant strain of E. coli, initially identified by an abnormally high frequency of recombination, has been found to be defective in the 5' -. 3' exonuclease associated with DNA polymerase I, but not in the polymerase activity. This defect is tolerated at 300, but is lethal at 43°. Like other polymerase I mutants, the strain is unusually sensitive to methyl methanesulfonate and to ultraviolet irradiation; it is also unable to support the growth of phage X defective in general recombination, and shows a reduced rate of joining of 10S "Okazaki fragments." These results demonstrate that a functional DNA polymerase I is essential for normal growth and viability in E. coli K12.Escherichia coli K12 strains defective in the structural gene for DNA polymerase I (polA) have been extensively described (1-5). These mutants are abnormally sensitive to ultraviolet irradiation and to the alkylating agent methyl methanesulfonate, and show retarded joining of the 10S "Okazaki fragments" (6)(7)(8). A recent analysis of partially purified fractions derived from..several of the polA mutants has demonstrated that although they contain much reduced levels of DNA polymerase I activity the 5' --3' exonuclease associated with the polymerase I is present in nearly normal amounts (9).We have found that polA mutants are also characterized by an increased frequency of recombination at least under some conditions (7). By screening mutagenized cells for this phenotype, we have isolated a novel polA mutant (polAexl) that we describe in this paper. This mutant has a normal level of polymerase I activity but a greatly reduced level of 5' -_ 3' exonuclease. Unlike other polA mutants, it is conditionally lethal. MATERIALS AND METHODSBacterial and Phage Strains. All bacterial strains are derived from E. coli K12. Their origin and genotypes are given in Table 1. Phage X defective in general recombination (A red 3) was provided by D. Freifelder.Microbiological Procedures. Pivir phage stocks were prepared and used for transduction, and Hfr crosses were performed as described by Miller (10). Sensitivity to ultraviolet irradiation was determined as follows. Cells grown at 30°in H broth (11) to A595 = 0.5 were chilled, harvested, and resuspended in an equal volume of chilled M63 medium (10); 10 ml of a 5-fold dilution of this suspension in a glass petri plate was irradiated (25 ergs per mm2 per sec) with a General Electric germicidal lamp; and samples were removed at various times, diluted in chilled H broth, and plated on yeast extract-tryptone plates (10) that were incubated at 300 in the dark. Sensitivity to methyl methanesulfonate was determined on fresh yeast extract-tryptone plates supplemented with 0.04% methyl methanesulfonate. Cultures were treated with ethyl methanesulfonate as described by Miller (10). Lactosetetrazolium indicator plates were prepared according to Miller (10). Ability to form plaques of phage A was determined on tryptone plates as described by Miller (10). Unless otherwise noted, E. coli polAexl strains were grown at ...

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Slow Joining of Newly Replicated DNA Chains in DNA Polymerase I-Deficient Escherichia coli Mutants

Cited by 143 publications

References 33 publications

The Replication Intermediates in Escherichia coli Are Not the Product of DNA Processing or Uracil Excision

The Replication Intermediates in Escherichia coli Are Not the Product of DNA Processing or Uracil Excision

RNA-Linked Nascent DNA Fragments in Escherichia coli

A Conditional Lethal Mutant of Escherichia coli K12 Defective in the 5′ → 3′ Exonuclease Associated with DNA Polymerase I

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