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...

We investigated the characteristics of a lambdoid prophage, nicknamed Lula, contaminating E. coli strains from several sources, that allowed it to spread horizontally in the laboratory environment. We found that new Lula infections are inconspicuous; at the same time, Lula lysogens carry unusually high titers of the phage in their cultures, making them extremely infectious. In addition, Lula prophage interferes with P1 phage development and induces its own lytic development in response to P1 infection, turning P1 transduction into an efficient vehicle of Lula spread. Thus, using Lula prophage as a model, we reveal the following principles of survival and reproduction in the laboratory environment: 1) stealth (via laboratory material commensality), 2) stability (via resistance to specific protocols), 3) infectivity (via covert yet aggressive productivity and laboratory protocol hitchhiking). Lula, which turned out to be identical to bacteriophage phi80, also provides an insight into a surprising persistence of T1-like contamination in BAC libraries.

Transcription of closed circular DNA templates in the presence of DNA gyrase is known to stimulate negative DNA supercoiling both in vivo and in vitro. It has proven elusive, however, to establish a general system in vitro that supports transcription-coupled DNA supercoiling (TCDS) by the "twin-domain" mechanism (Liu, L. F. and Wang, J. C. (1987) Proc. Natl. Acad. Sci. USA 84, 7024 -7027) that operates in bacteria. In this report, we examine the properties of TCDS in defined protein systems that minimally contained T7 RNA polymerase and DNA gyrase. Specifically designed plasmid DNA templates permitted us to control the location and length of RNA transcripts. We demonstrate that TCDS takes place by two separate, and apparently independent, mechanistic pathways in vitro. The first supercoiling pathway, which is not likely to be significant in vivo, was found to be dependent on R-loop formation and could be suppressed by the presence of RNase H or bacterial HU protein. The second pathway for TCDS was much more potent, but became predominant in vitro only when sequence-specific DNA-bending proteins were present during transcription, and RNA transcript lengths exceeded 3 kb. This major supercoiling route was shown to be resistant to RNase H and had functional properties consistent with those predicted for the twin-domain mechanism. For example, DNA supercoiling activity was proportional to RNA transcript length and was greatly stimulated by macromolecular crowding agents. Under optimal conditions, the twin domain pathway of TCDS rapidly and efficiently generated superhelicity levels more than twice that typically found in vivo.DNA transactions in prokaryotic cells, such as DNA replication, transcription, and recombination, are often dependent on or stimulated by negative supercoiling of the DNA template (1-3). For most of these instances, the available evidence indicates that negative superhelical tension facilitates an early step in the reaction pathway, typically, opening of the DNA double helix. The prokaryotic type II DNA topoisomerase, DNA gyrase, is the enzyme responsible for introducing negative supercoils into bacterial chromosomes and plasmids (4 -6). While none of the eukaryotic DNA topoisomerases is capable of negatively supercoiling DNA, it is now apparent that some enzymes that translocate processively along DNA, such as RNA polymerase, can affect DNA superhelical tension at a local level (7-10). These localized changes in DNA supercoiling can be used to drive or regulate DNA transactions at nearby sites.Liu and Wang (8) proposed an elegant model to explain how transcription by RNA polymerase can be used to stimulate DNA supercoiling. This model, termed the twin supercoil domain model, has received broad experimental support in studies of DNA supercoiling in living cells. The twin-domain model postulates that rotation of the RNA polymerase-RNA complex around the DNA helical axis during transcription becomes increasingly difficult as the size of the growing RNA chain increases. At a critical juncture, it ...

Chromosomal DNA replication intermediates, revealed in ligase-deficient conditions in vivo, are of low molecular weight independently of the organism, suggesting discontinuous replication of both the leading and the lagging DNA strands. Yet, in vitro experiments with purified enzymes replicating sigma-structured substrates show continuous synthesis of the leading DNA strand in complete absence of ligase, supporting the textbook model of semi-discontinuous DNA replication. The discrepancy between the in vivo and in vitro results is rationalized by proposing that various excision repair events nick continuously-synthesized leading strands after synthesis, producing the observed low molecular weight intermediates. Here we show that, in an E. coli ligase-deficient strain with all known excision repair pathways inactivated, new DNA is still synthesized discontinuously. Furthermore, hybridization to strand-specific targets demonstrates that the low molecular weight replication intermediates come from both the lagging and the leading strands. These results support the model of discontinuous leading strand synthesis in E. coli.

Phenol-chloroform extraction of [32 P]orthophosphate-labeled Escherichia coli cells followed by alkaline gel electrophoresis revealed, besides the expected chromosomal DNA, two non-DNA species that we have identified as lipopolysaccharides and polyphosphates by using a combination of biochemical and genetic techniques. We used this serendipitously found straightforward protocol for direct polyphosphate detection to quantify polyphosphate levels in E. coli mutants with diverse defects in the DNA metabolism. We detected increased polyphosphate accumulation in the ligA, ligA recBCD, dut ung, and thyA mutants. Polyphosphate accumulation may thus be an indicator of general DNA stress.DNA replication intermediates, also known as Okazaki fragments, have classically been detected by pulse labeling thymine-limited thyA mutant cells with [ 3 H]thymidine, a DNAspecific label (27). However, when limited for thymidine, thyA mutants are known to undergo thymine-less death (1), a phenomenon during which chromosomal DNA suffers singlestrand breaks (24). (10,15,30).To avoid the possibility of thymine starvation in our experiments, we also attempted to visualize Okazaki fragments by using the [ 32 P]orthophosphate label which we routinely employ to label chromosomal DNA for pulsed-field gel electrophoresis (17, 36). Since we expected that the bulk of the 32 P label will be deposited into RNA, we removed RNA altogether by separating chromosomal DNA from replication intermediates in alkaline agarose gels. We found, however, that Okazaki pieces cannot be detected using [ 32 P]orthophosphate even by alkaline agarose because there are other molecules in larger amounts in the cells that take in 32 P-label and mask the replication intermediates. We report on the identification and quantification of two of the "masking species" in wild-type Escherichia coli, as well as in several mutants. MATERIALS AND METHODSBacterial strains, growth conditions, labeling, and isolation of phosphatecontaining species. All strains are described in Table 1. Cells were grown with shaking at 30°C in MOPS (morpholinepropanesulfonic acid) low-phosphate medium (25) supplemented with 0.2% Casamino Acids to an optical density at 600 nm (OD 600 ) of 0.2 to 0.4. Cultures were further incubated at 30°C, 37°C, or 42°C and labeled with [ 32 P]orthophosphate (5 to 10 Ci/ml) for the amount of time indicated in the figures. Samples were processed by spinning down the cells and resuspending them in 50 l of 20% sucrose in Tris-EDTA. Three hundred fifty microliters of 2% sodium dodecyl sulfate (SDS) were added, and after thorough mixing, the cells were lysed by incubation at 70°C for 10 min. Isolation of nucleic acids was achieved by subsequent extraction with 400 l of phenol, followed by 400 l of phenol-chloroform, and finally, 400 l of chloroform, with two ethanol precipitations (18). Alternatively, the Wizard genomic DNA purification kit (Promega) was used.Enzymatic reactions. DNase I, exonuclease I, and calf intestinal phosphatase (CIP) were purchased from New England ...