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

Abstract

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...

Gaps and forks in DNA replication: Rediscovering old models

“…This issue has recently been re-addressed by Amado and Kuzminov, who, using a temperature-sensitive ligase mutant of E. coli, showed that essentially all newly synthesised DNA was synthesised in small pieces [17]. This strongly suggests that both leading and lagging strands can be synthesised discontinuously in vivo, contrary to accepted dogma, but supporting the original model [13].…”

ThemutTDefect Does Not Elevate Chromosomal Fragmentation inEscherichia coliBecause of the Surprisingly Low Levels of MutM/MutY-Recognized DNA Modifications

“…Next, we considered the possibility that base analogs are incorporated into the DNA of mutT mutants and are then excised, but this excision is somehow different from the excision in dut or rdgB mutants in that it does not cause chromosomal fragmentation. In dut mutant strains, the high level of uracil misincorporation combined with the efficient excision repair elevates the steady-state level of nicks in DNA (1,43,76). Nicks can be detected as relaxation of supercoiled plasmids.…”

“…On the other hand, if uracil forms within DNA as a result of cytosine deamination (52), uracil DNA glycosylase (UDG), with the help of exonuclease III, acts to remove this base (50,75). Interestingly, UDG does not distinguish between the U · A base pairs (the products of uracil incorporation) and the U · G base pairs (the products of cytosine deamination), excising both uracils equally well (51,63), which leads to the elevated frequency of excision repair intermediates (nicks) in the dut mutants of E. coli (1,43,76).…”

Synthetic Lethality with the dut Defect in Escherichia coli Reveals Layers of DNA Damage of Increasing Complexity Due to Uracil Incorporation

“…However, no uracil was detected in the bulk DNA of wild-type or dut-1 mutant cells (35), due to the presence of a highly active enzyme, UDG (42) (the product of the ung gene [17]). UDG excises every uracil-DNA almost as soon as it is incorporated; this excision generates the short replication intermediates in the dut mutants (1,74). In fact, it is the uracil excision that eventually kills, directly or indirectly, SL(dut) mutants, as all of them are known to be suppressed by ung inactivation (34,70,73).…”