Size Distribution and Molecular Polarity of Newly Replicated DNA in
            <i>Escherichia coli</i>

Louarn, Jean-Michel; Bird, Robert E.

doi:10.1073/pnas.71.2.329

Cited by 30 publications

(17 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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.…”

supporting

confidence: 51%

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

supporting

confidence: 51%

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

“…Several groups have reported that they never observe more than 50% of the acid precipitable radioactivity in the Okazaki peak (Painter & Schaefer, 1969;Hyodo et al, 1970Hyodo et al, , 1971Iyer & Lark, 1970;Eisenberg & Dernhardt, 1974;Francke & Hunter, 1974a;Friedman, 1974;Louarn & Bird, 1974). SOIDe of the reports are not convincing because pulse-times were so long that the percentage of the total acid precipitable radioactivity found in the Okazaki peak is probably misleadingly small (Hyodo et al, 1970;.…”

Section: Are Both Sides Nlade Discontinuously?mentioning

confidence: 82%

“…As I will point Qut in the Discussion section, this is not an easy question to resolve. It is made even more difficult by the fact that the 5'----,)l3' strand may be replicated more or less discontinuously depending on mutations (Lark, 1972;Louarn & Bird, 1974) or on other conditions affecting the rate of synthesis (Herrmann et al, 1972;Olivera & Bonhoeffer, 1972). …”

Section: Are Both Strands Made Discontinuously?mentioning

confidence: 99%

“…, 1975;Tseng & Goulian, 1975a). Others observe less than 500/0 of the radioactivity in the small pieces even after short pulses, and conclude that only one strand is made discontinuously (Painter & Schaefer, 1969;Hyodo et a1., 1970Hyodo et a1., , 1971Iyer and Lark, 1970;Eisenberg & Dernhardt, 1974;Francke & Hunter, 1974a;Friedman, 1974;Louarn & Bird, 1974).…”

Section: Are Both Strands Made Discontinuously?mentioning

confidence: 99%

See 1 more Smart Citation

Discontinuous DNA Synthesis in Mammalian Cells

Huberman¹,

Horwitz²

1974

Cold Spring Harbor Symposia on Quantitative Biology

View full text Add to dashboard Cite

“…The direction of replication across a given locus may be determined from the strand preference of Okazaki fragments specific for the marker considered, a technique well suited to the case of X prophages (23). Strains carrying XTSK prophages at zcjlS2 or dif positions, with or without psrA* (Fig.…”

Section: Resultsmentioning

confidence: 99%

Hyperrecombination in the terminus region of the Escherichia coli chromosome: possible relation to nucleoid organization

et al. 1994

View full text Add to dashboard Cite

The terminus region of the Escherichia coli chromosome is the scene of frequent homologous recombination. This can be demonstrated by formation of deletions between directly repeated sequences which flank a genetic marker whose loss can be easily detected. We report here that terminal recombination events are restricted to a relatively large terminal recombination zone (TRZ). On one side of the TRZ, the transition from the region with a high excision rate to the normal (low) excision rates of the rest of the chromosome occurs along a DNA stretch of less than 1 min. No specific border of this domain has been defined. To identify factors inducing terminal recombination, we examined its relation to two other phenomena affecting the same region, site-specific recombination at the dif locus and site-specific replication pausing. Both the location and the efficiency of terminal recombination remained unchanged after inactivation of the dif-specific recombination system. Similarly, inactivation of site-specific replication pausing or displacement of the replication fork trap so that termination occurs about 200 kb away from the normal region had no clear effect on this phenomenon.Therefore, terminal recombination is not a direct consequence of either dif-specific recombination or replication termination. Furthermore, deletions encompassing the wild-type TRZ do not eliminate hyperrecombination. Terminal recombination therefore cannot be attributed to the activity of some unique sequence of the region. A possible explanation of terminal hyperrecombination involves nucleoid organization and its remodeling after replication: we propose that postreplicative reconstruction of the nucleoid organization results in a displacement of the catenation links between sister chromosomes to the last chromosomal domain to be rebuilt. Unrelated to replication termination, this process would facilitate interactions between the catenated molecules and would make the domain highly susceptible to recombination between sister chromosomes.The region of the Escherichia coli chromosome in which replication terminates is implicated in two recombination events occurring at high frequencies in cells with a wild-type cell cycle machinery. One is site-specific recombination, controlled by the XerCD recombinases (4), which takes place at a unique site, dif, located at 34 min on the genetic map (3,5,20).The dif-specific recombination probably participates in chromosome dimer resolution, since bacteria deleted for the dif locus, or made XerC-, have a tendency to form filaments (3,20), but the exact physiological role of dif-mediated recombination is not precisely known. The other very frequent event is homologous recombination between directly repeated sequences (24). The position where the phenomenon was the most striking (the zdd263::TnS locus) maps between dif and the first replication terminator acting on forks moving rightward in Fig. 1B, terC pothesis concerning the mechanism and the role for the hyperrecombination in the terminus region has been...

show abstract

Size Distribution and Molecular Polarity of Newly Replicated DNA in Escherichia coli

Cited by 30 publications

References 28 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

Discontinuous DNA Synthesis in Mammalian Cells

Hyperrecombination in the terminus region of the Escherichia coli chromosome: possible relation to nucleoid organization

Contact Info

Product

Resources

About