Nutritional conditions and oxygen concentration affect spontaneous occurrence of homologous recombination events but not spontaneous mutagenesis in Escherichia coli.

Effects of environmental factors for growth of Escherichia coli on spontaneous mutagenesis and homologous recombination events are described. By analyzing rifampicin-resistant (Rifr) mutation frequencies in an E. coli strain lacking MutM and MutY repair enzymes, which suppress base substitution mutations caused by 8-oxoguanine (7,8 dihydro-8-oxoguanine; 8-oxoG) in DNA, we examined levels of oxidative DNA damage produced in normally growing cells. The level of 8-oxoG DNA damage was about 9- and 63-fold higher in cells grown in M9-glucose and M9-glycerol media, respectively, than in those grown in LB medium. We also found that about 14-fold more 8-oxoG DNA damage was produced in cells grown in about 0.1% oxygen than in those grown in the normal atmosphere. However, Rifr mutation frequency in wild-type cells was unchanged in such different growth conditions, suggesting that the capacity of repair mechanisms is sufficient to suppress mutations caused by 8-oxoG even at very high levels in cells growing in the particular conditions. On the other hand, the frequency of spontaneous homologous recombination events in wild-type E. coli cells varied with different growth conditions. When cells were grown in M9-glucose and M9-glycerol media, the spontaneous recombination frequency increased to about 4.3- and 7.3-fold, respectively, higher than that in LB medium. Likewise, the spontaneous recombination frequency was about 3.5-fold higher in cells growing in the hypoxic condition than in cells growing in the atmosphere. When cells were grown in anaerobic conditions, the recombination frequency decreased to half of that in the atmosphere. These data indicated that spontaneous homologous recombination is highly responsive to environmental factors such as nutrition and oxygen concentration.

Replication fork progression is paused in two large chromosomal zones flanking the DNA replication origin in Escherichia coli.

Although the speed of nascent DNA synthesis at individual replication forks is relatively uniform in bacterial cells, the dynamics of replication fork progression on the chromosome are hampered by a variety of natural impediments. Genome replication dynamics can be directly measured from an exponentially growing cell population by sequencing newly synthesized DNA strands that were specifically pulse-labeled with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU). However, a short pulse labeling with BrdU is impracticable for bacteria because of poor incorporation of BrdU into the cells, and thus, the genomewide dynamics of bacterial DNA replication remain undetermined. Using a new thymidine-requiring Escherichia coli strain, eCOMB, and high-throughput sequencing, we succeeded in determining the genomewide replication profile in bacterial cells. We also found that fork progression is paused in two ~200-kb chromosomal zones that flank the replication origin in the growing cells. This origin-proximal obstruction to fork progression was overcome by an increased thymidine concentration in the culture medium and enhanced by inhibition of transcription. These indicate that DNA replication near the origin is sensitive to the impediments to fork progression, namely a scarcity of the DNA precursor deoxythymidine triphosphate and probable conflicts between replication and transcription machineries.

Recombinase and translesion DNA polymerase decrease the speed of replication fork progression during the DNA damage response in Escherichia coli cells.

The SOS response is a DNA damage response pathway that serves as a general safeguard of genome integrity in bacteria. Extensive studies of the SOS response in Escherichia coli have contributed to establishing the key concepts of cellular responses to DNA damage. However, how the SOS response impacts on the dynamics of DNA replication fork movement remains unknown. We found that inducing the SOS response decreases the mean speed of individual replication forks by 30-50% in E. coli cells, leading to a 20-30% reduction in overall DNA synthesis. dinB and recA belong to a group of genes that are upregulated during the SOS response, and encode the highly conserved proteins DinB (also known as DNA polymerase IV) and RecA, which, respectively, specializes in translesion DNA synthesis and functions as the central recombination protein. Both genes were independently responsible for the SOS-dependent slowdown of replication fork progression. Furthermore, fork speed was reduced when each gene was ectopically expressed in SOS-uninduced cells to the levels at which they are expressed in SOS-induced cells. These results clearly indicate that the increased expression of dinB and recA performs a novel role in restraining the progression of an unperturbed replication fork during the SOS response.

DNA polymerase IV mediates efficient and quick recovery of replication forks stalled at N2-dG adducts.

Escherichia coli DNA polymerase IV (Pol IV, also known as DinB) is a Y-family DNA polymerase capable of catalyzing translesion DNA synthesis (TLS) on certain DNA lesions, and accumulating data suggest that Pol IV may play an important role in copying various kinds of spontaneous DNA damage including N(2)-dG adducts and alkylated bases. Pol IV has a unique ability to coexist with Pol III on the same ß clamp and to positively dissociate Pol III from ß clamp in a concentration-dependent manner. Reconstituting the entire process of TLS in vitro using E. coli replication machinery and Pol IV, we observed that a replication fork stalled at (-)-trans-anti-benzo[a]pyrene-N(2)-dG lesion on the leading strand was efficiently and quickly recovered via two sequential switches from Pol III to Pol IV and back to Pol III. Our results suggest that TLS by Pol IV smoothes the way for the replication fork with minimal interruption.

A single-molecule approach to DNA replication in Escherichia coli cells demonstrated that DNA polymerase III is a major determinant of fork speed.

The replisome catalyses DNA synthesis at a DNA replication fork. The molecular behaviour of the individual replisomes, and therefore the dynamics of replication fork movements, in growing Escherichia coli cells remains unknown. DNA combing enables a single-molecule approach to measuring the speed of replication fork progression in cells pulse-labelled with thymidine analogues. We constructed a new thymidine-requiring strain, eCOMB (E. coli for combing), that rapidly and sufficiently incorporates the analogues into newly synthesized DNA chains for the DNA-combing method. In combing experiments with eCOMB, we found the speed of most replication forks in the cells to be within the narrow range of 550-750 nt s(-1) and the average speed to be 653 ± 9 nt s(-1) (± SEM). We also found the average speed of the replication fork to be only 264 ± 9 nt s(-1) in a dnaE173-eCOMB strain producing a mutant-type of the replicative DNA polymerase III (Pol III) with a chain elongation rate (300 nt s(-1) ) much lower than that of the wild-type Pol III (900 nt s(-1) ). This indicates that the speed of chain elongation by Pol III is a major determinant of replication fork speed in E. coli cells.

Quick replication fork stop by overproduction of Escherichia coli DinB produces non-proliferative cells with an aberrant chromosome.

Escherichia coli dinB encodes the translesion DNA polymerase DinB, which can inhibit progression of replication forks in a dose-dependent manner, independent of exogenous DNA damage. We reported previously that overproduction of DinB from a multicopy dinB plasmid immediately abolished ongoing replication fork progression, and the cells rapidly and drastically lost colony-forming ability, although the mechanisms underlying this lethality by severe replication fork stress remained unclear. Here, we show that the reduced colony-forming ability in the dinB-overexpressing cells is independent of the specific toxin genes that trigger programmed bacterial cell death when replication is blocked by depletion of the dNTP pool. After DinB abolished replication fork progression and colony-forming ability, most of the cells were still viable, as judged by fluorescent dye staining, but contained irregularly shaped nucleoids in which chromosomal DNA was preferentially lost in the replication terminus region relative to the replication origin region. Flow cytometric analysis of the cells revealed chromosomal damage and the eventual appearance of cell populations with less than single-chromosome DNA content, reminiscent of sub-G1 cells with lethal DNA content produced during eukaryotic apoptosis. This reduced DNA content was not observed after replication fork progression was quickly stopped in temperature-sensitive dnaB helicase mutant cells at a non-permissive temperature. Thus, the quick replication stop provoked by excess DinB uniquely generates temporarily viable but non-reproductive cells possessing a fatally depleted chromosomal content, which may represent one of the possible fates of an E. coli cell whose replication is overwhelmingly compromised.

Escherichia coli DinB inhibits replication fork progression without significantly inducing the SOS response.

The SOS response is readily triggered by replication fork stalling caused by DNA damage or a dysfunctional replicative apparatus in Escherichia coli cells. E. coli dinB encodes DinB DNA polymerase and its expression is upregulated during the SOS response. DinB catalyzes translesion DNA synthesis in place of a replicative DNA polymerase III that is stalled at a DNA lesion. We showed previously that DNA replication was suppressed without exogenous DNA damage in cells overproducing DinB. In this report, we confirm that this was due to a dose-dependent inhibition of ongoing replication forks by DinB. Interestingly, the DinB-overproducing cells did not significantly induce the SOS response even though DNA replication was perturbed. RecA protein is activated by forming a nucleoprotein filament with single-stranded DNA, which leads to the onset of the SOS response. In the DinB-overproducing cells, RecA was not activated to induce the SOS response. However, the SOS response was observed after heat-inducible activation in strain recA441 (encoding a temperature-sensitive RecA) and after replication blockage in strain dnaE486 (encoding a temperature-sensitive catalytic subunit of the replicative DNA polymerase III) at a non-permissive temperature when DinB was overproduced in these cells. Furthermore, since catalytically inactive DinB could avoid the SOS response to a DinB-promoted fork block, it is unlikely that overproduced DinB takes control of primer extension and thus limits single-stranded DNA. These observations suggest that DinB possesses a feature that suppresses DNA replication but does not abolish the cell's capacity to induce the SOS response. We conclude that DinB impedes replication fork progression in a way that does not activate RecA, in contrast to obstructive DNA lesions and dysfunctional replication machinery.

Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA.

DNA polymerase IV (Pol IV) is one of three translesion polymerases in Escherichia coli. A mass spectrometry study revealed that single-stranded DNA-binding protein (SSB) in lysates prepared from exponentially-growing cells has a strong affinity for column-immobilized Pol IV. We found that purified SSB binds directly to Pol IV in a pull-down assay, whereas SSBΔC8, a mutant protein lacking the C-terminal tail, failed to interact with Pol IV. These results show that the interaction between Pol IV and SSB is mediated by the C-terminal tail of SSB. When polymerase activity was tested on an SSBΔC8-coated template, we observed a strong inhibition of Pol IV activity. Competition experiments using a synthetic peptide containing the amino acid sequence of SSB tail revealed that the chain-elongating capacity of Pol IV was greatly impaired when the interaction between Pol IV and SSB tail was inhibited. These results demonstrate that Pol IV requires the interaction with the C-terminal tail of SSB to replicate DNA efficiently when the template ssDNA is covered with SSB. We speculate that at the primer/template junction, Pol IV interacts with the tail of the nearest SSB tetramer on the template, and that this interaction allows the polymerase to travel along the template while disassembling SSB.