A unique property of the replicating region of chromosomal DNA

Sakabe, K.; Okazaki, Reiji

doi:10.1016/0005-2787(66)90088-8

Cited by 103 publications

(45 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These experimental results were confirmed by others (6, 12), and the LMW replication intermediates became known as "Okazaki fragments. "The main result of these early studies was that all new DNA, on both the lagging and the leading strands, appeared first in small pieces (1,5,8). This interpretation was confirmed in polA mutants, deficient in DNA polymerase I (13-15) and with DNA ligase mutants (11,16,17).…”

mentioning

confidence: 86%

“…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).…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Amado

Kuzminov

2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

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...

show abstract

mentioning

confidence: 86%

mentioning

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

View full text Add to dashboard Cite

show abstract

“…These segments, originally found by Sakabe and Okazaki (7) in replicating cells of Escherichia coli, and since found to be associated with replication in other cells, appear to be intermediates in replication. Recently, Okazaki and associates have characterized them with respect to size, direction of chain growth, and rate of joining by ligase in normal-as well as polymerase I-deficient-strains (8,9).…”

mentioning

confidence: 77%

Replication of DNA in Mammalian Chromosomes: Isolation of Replicating Segments

Taylor

1973

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

View full text Add to dashboard Cite

From Chinese hamster cells, blocked at the beginning of the S phase by treatment with fluorodeoxyuridine and pulse-labeled with [3Hithyzuidine, one can release segments of native DNA containing template polynucleotide chains and the most recently replicated DNA. The segments, which can be released without shear by Iysis at temperatures below the melting point for the double helix, can be separated from both the nascent single chains (Okazaki pieces) and the long strands of native DNA in sodium perchlorate gradients. These 26S segments are equivalent to about 6 pm of native DNA, but may be replication forks rather than linear pieces.A highly regulated nuclease activity coupled with a ligase activity would appear to be necessary for the unwinding of [3H]thymidine during the subsequent S phase. After a pulselabel of 20 min, when most growing chains of [3HIDNA were long enough to sediment well ahead of the short 14C-labeled template segments, their presence and distinctive size could be determined in alkaline sucrose gradients (2). There was a clear size discontinuity between the template segments and the major component of nuclear DNA, which was dissociated into segments more than 100 Am in length. The role of such fragments in replication could only be inferred, since no direct evidence of a connection between these 2-pm fragments and the much smaller fragments of nascent DNA could be established (3). The nascent, single-chain DNA from mammalian cells is estimated to be 0.3-0.5 pm in length (4-6).These segments, originally found by Sakabe and Okazaki (7) in replicating cells of Escherichia coli, and since found to be associated with replication in other cells, appear to be intermediates in replication. Recently, Okazaki and associates have characterized them with respect to size, direction of chain growth, and rate of joining by ligase in normal-as well as polymerase I-deficient-strains (8, 9). * This paper is no. I in a series. (12) were grown (13) in Ham's F-10 medium. Some advantages of these cells for studying DNA replication are their fast growth (GI = 5.5 hr, S = 6.5 hr, and G2 + M = 3.5 hr) under our culture conditions at 36-370 and their rather firm attachment to the substrate except when dividing. Cells were grown in roller bottles with a diameter of about 10 cm (Bellco). When rotated at about 0.5 rpm, the dividing cells may detach temporarily, but reattach early in G1. However, when rotated at 3 rpm, most dividing cells cannot reattach and one may collect several million dividing cells in an hour from each culture of 1 to 2 X 10' cells.The dividing cells were transferred to polystyrene flasks and, after firm attachment (about 3 hr), the medium was exchanged for Ham's medium without thymidine and with 1 pAM fluorodeoxyuridine (FdU) to inhibit further synthesis of thymidylate. Dialyzed serum was also used in this medium to reduce the supply of exogenous thymidine. In spite of these precautions, the cells appear to have enough thymidine to replicate 5-10% of the DNA before the block becomes effect...

show abstract

“…In those cases, the leading strand usually displayed a positive GC skew, or sometimes a TA skew, while the lagging strand is the opposite. The bias in base composition is closely related with the asymmetric replication machinery between the two strands: the leading strand replicates continuously, whereas the lagging strand replicates in short fragments, forming Okazaki fragments (Okazaki et al, 1967;Sakabe & Okazaki, 1966). The different replication mechanisms may exert differential mutation or selection pressures on the two strands and, thus, contribute to the biased base composition (Rocha, 2002;Tillier & Collins, 2000a).…”

Section: Introductionmentioning

confidence: 99%

Novel genomic rearrangements mediated by multiple genetic elements in Streptococcus pyogenes M23ND confer potential for evolutionary persistence

et al. 2016

View full text Add to dashboard Cite

Symmetric genomic rearrangements around replication axes in genomes are commonly observed in prokaryotic genomes, including Group A Streptococcus (GAS). However, asymmetric rearrangements are rare. Our previous studies showed that the hypervirulent invasive GAS strain, M23ND, containing an inactivated transcriptional regulator system, covRS, exhibits unique extensive asymmetric rearrangements, which reconstructed a genomic structure distinct from other GAS genomes. In the current investigation, we identified the rearrangement events and examined the genetic consequences and evolutionary implications underlying the rearrangements. By comparison with a close phylogenetic relative, M18-MGAS8232, we propose a molecular model wherein a series of asymmetric rearrangements have occurred in M23ND, involving translocations, inversions and integrations mediated by multiple factors, viz., rRNA-comX (factor for late competence), transposons and phage-encoded gene segments. Assessments of the cumulative gene orientations and GC skews reveal that the asymmetric genomic rearrangements did not affect the general genomic integrity of the organism. However, functional distributions reveal re-clustering of a broad set of CovRS-regulated actively transcribed genes, including virulence factors and metabolic genes, to the same leading strand, with high confidence (p-value~10 À10 ). The re-clustering of the genes suggests a potential selection advantage for the spatial proximity to the transcription complexes, which may contain the global transcriptional regulator, CovRS, and other RNA polymerases. Their proximities allow for efficient transcription of the genes required for growth, virulence and persistence. A new paradigm of survival strategies of GAS strains is provided through multiple genomic rearrangements, while, at the same time, maintaining genomic integrity.

show abstract

A unique property of the replicating region of chromosomal DNA

Cited by 103 publications

References 9 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

Replication of DNA in Mammalian Chromosomes: Isolation of Replicating Segments

Novel genomic rearrangements mediated by multiple genetic elements in Streptococcus pyogenes M23ND confer potential for evolutionary persistence

Contact Info

Product

Resources

About