Browse
Submit Data
Databases
API
Help

Dataset Information

23 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Spontaneous DNA breakage in single living Escherichia coli cells.

ABSTRACT: Spontaneous DNA breakage is predicted to be a frequent, inevitable consequence of DNA replication and is thought to underlie much of the genomic change that fuels cancer and evolution. Despite its importance, there has been little direct measurement of the amounts, types, sources and fates of spontaneous DNA lesions in living cells. We present a direct, sensitive flow cytometric assay in single living Escherichia coli cells for DNA lesions capable of inducing the SOS DNA damage response, and we report its use in quantification of spontaneous DNA double-strand breaks (DSBs). We report efficient detection of single chromosomal DSBs and rates of spontaneous breakage approximately 20- to 100-fold lower than predicted. In addition, we implicate DNA replication in the origin of spontaneous DSBs with the finding of fewer spontaneous DSBs in a mutant with altered DNA polymerase III. The data imply that spontaneous DSBs induce genomic changes and instability 20-100 times more potently than previously appreciated. Finally, FACS demonstrated two main cell fates after spontaneous DNA damage: viability with or without resumption of proliferation.

SUBMITTER: Pennington JM

PROVIDER: S-EPMC2856310 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Heterogeneity of spontaneous DNA replication errors in single isogenic <i>Escherichia coli</i> cells.

Project description:Despite extensive knowledge of the molecular mechanisms that control mutagenesis, it is not known how spontaneous mutations are produced in cells with fully operative mutation-prevention systems. By using a mutation assay that allows visualization of DNA replication errors and stress response transcriptional reporters, we examined populations of isogenic Escherichia coli cells growing under optimal conditions without exogenous stress. We found that spontaneous DNA replication errors in proliferating cells arose more frequently in subpopulations experiencing endogenous stresses, such as problems with proteostasis, genome maintenance, and reactive oxidative species production. The presence of these subpopulations of phenotypic mutators is not expected to affect the average mutation frequency or to reduce the mean population fitness in a stable environment. However, these subpopulations can contribute to overall population adaptability in fluctuating environments by serving as a reservoir of increased genetic variability.

| S-EPMC6010332 | biostudies-literature

Single-molecule imaging of DNA gyrase activity in living Escherichia coli.

Project description:Bacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription. Removal of these positive supercoils is essential for replication fork progression and for the overall unlinking of the two duplex DNA strands, as well as for ongoing transcription. To address how gyrase copes with these topological challenges, we used high-speed single-molecule fluorescence imaging in live Escherichia coli cells. We demonstrate that at least 300 gyrase molecules are stably bound to the chromosome at any time, with ?12 enzymes enriched near each replication fork. Trapping of reaction intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell times of ?2 s were observed for the dispersed gyrase molecules, which we propose maintain steady-state levels of negative supercoiling of the chromosome. In contrast, the dwell time of replisome-proximal molecules was ?8 s, consistent with these catalyzing processive positive supercoil relaxation in front of the progressing replisome.

| S-EPMC6326794 | biostudies-literature

Engineered proteins detect spontaneous DNA breakage in human and bacterial cells.

Project description:Spontaneous DNA breaks instigate genomic changes that fuel cancer and evolution, yet direct quantification of double-strand breaks (DSBs) has been limited. Predominant sources of spontaneous DSBs remain elusive. We report synthetic technology for quantifying DSBs using fluorescent-protein fusions of double-strand DNA end-binding protein, Gam of bacteriophage Mu. In Escherichia coli GamGFP forms foci at chromosomal DSBs and pinpoints their subgenomic locations. Spontaneous DSBs occur mostly one per cell, and correspond with generations, supporting replicative models for spontaneous breakage, and providing the first true breakage rates. In mammalian cells GamGFP-labels laser-induced DSBs antagonized by end-binding protein Ku; co-localizes incompletely with DSB marker 53BP1 suggesting superior DSB-specificity; blocks resection; and demonstrates DNA breakage via APOBEC3A cytosine deaminase. We demonstrate directly that some spontaneous DSBs occur outside of S phase. The data illuminate spontaneous DNA breakage in E. coli and human cells and illustrate the versatility of fluorescent-Gam for interrogation of DSBs in living cells. DOI:http://dx.doi.org/10.7554/eLife.01222.001.

| S-EPMC3809393 | biostudies-literature

Single-molecule imaging of UvrA and UvrB recruitment to DNA lesions in living Escherichia coli.

Project description:Nucleotide excision repair (NER) removes chemically diverse DNA lesions in all domains of life. In Escherichia coli, UvrA and UvrB initiate NER, although the mechanistic details of how this occurs in vivo remain to be established. Here, we use single-molecule fluorescence imaging to provide a comprehensive characterization of the lesion search, recognition and verification process in living cells. We show that NER initiation involves a two-step mechanism in which UvrA scans the genome and locates DNA damage independently of UvrB. Then UvrA recruits UvrB from solution to the lesion. These steps are coordinated by ATP binding and hydrolysis in the 'proximal' and 'distal' UvrA ATP-binding sites. We show that initial UvrB-independent damage recognition by UvrA requires ATPase activity in the distal site only. Subsequent UvrB recruitment requires ATP hydrolysis in the proximal site. Finally, UvrA dissociates from the lesion complex, allowing UvrB to orchestrate the downstream NER reactions.

| S-EPMC5007444 | biostudies-literature

DNA polymerase switching: effects on spontaneous mutagenesis in Escherichia coli.

Project description:Escherichia coli possesses five known DNA polymerases (pols). Pol III holoenzyme is the cell's main replicase, while pol I is responsible for the maturation of Okazaki fragments and filling gaps generated during nucleotide excision repair. Pols II, IV and V are significantly upregulated as part of the cell's global SOS response to DNA damage and under these conditions, may alter the fidelity of DNA replication by potentially interfering with the ability of pols I and III to complete their cellular functions. To test this hypothesis, we determined the spectrum of rpoB mutations arising in an isogenic set of mutL strains differentially expressing the chromosomally encoded pols. Interestingly, mutagenic hot spots in rpoB were identified that are susceptible to the actions of pols I-V. For example, in a recA730 lexA(Def) mutL background most transversions were dependent upon pols IV and V. In contrast, transitions were largely dependent upon pol I and to a lesser extent, pol III. Furthermore, the extent of pol I-dependent mutagenesis at one particular site was modulated by pols II and IV. Our observations suggest that there is considerable interplay among all five E. coli polymerases that either reduces or enhances the mutagenic load on the E. coli chromosome.

| S-EPMC2680738 | biostudies-literature

Transforming DNA uptake gene orthologs do not mediate spontaneous plasmid transformation in Escherichia coli.

Project description:Spontaneous plasmid transformation of Escherichia coli occurs on nutrient-containing agar plates. E. coli has also been reported to use double-stranded DNA (dsDNA) as a carbon source. The mechanism(s) of entry of exogenous dsDNA that allows plasmid establishment or the use of DNA as a nutrient remain(s) unknown. To further characterize plasmid transformation, we first documented the stimulation of transformation by agar and agarose. We provide evidence that stimulation is not due to agar contributing a supplement of Ca(2+), Fe(2+), Mg(2+), Mn(2+), or Zn(2+). Second, we undertook to inactivate the E. coli orthologues of Haemophilus influenzae components of the transformation machine that allows the uptake of single-stranded DNA (ssDNA) from exogenous dsDNA. The putative outer membrane channel protein (HofQ), transformation pseudopilus component (PpdD), and transmembrane pore (YcaI) are not required for plasmid transformation. We conclude that plasmid DNA does not enter E. coli cells as ssDNA. The finding that purified plasmid monomers transform E. coli with single-hit kinetics supports this conclusion; it establishes that a unique monomer molecule is sufficient to give rise to a transformant, which is not consistent with the reconstitution of an intact replicon through annealing of partially overlapping complementary ssDNA, taken up from two independent monomers. We therefore propose that plasmid transformation involves internalization of intact dsDNA molecules. Our data together, with previous reports that HofQ is required for the use of dsDNA as a carbon source, suggest the existence of two routes for DNA entry, at least across the outer membrane of E. coli.

| S-EPMC2632100 | biostudies-literature

R-loops and nicks initiate DNA breakage and genome instability in non-growing Escherichia coli.

Project description:Double-stranded DNA ends, often from replication, drive genomic instability, yet their origin in non-replicating cells is unknown. Here we show that transcriptional RNA/DNA hybrids (R-loops) generate DNA ends that underlie stress-induced mutation and amplification. Depleting RNA/DNA hybrids with overproduced RNase HI reduces both genomic changes, indicating RNA/DNA hybrids as intermediates in both. An Mfd requirement and inhibition by translation implicate transcriptional R-loops. R-loops promote instability by generating DNA ends, shown by their dispensability when ends are provided by I-SceI endonuclease. Both R-loops and single-stranded endonuclease TraI are required for end formation, visualized as foci of a fluorescent end-binding protein. The data suggest that R-loops prime replication forks that collapse at single-stranded nicks, producing ends that instigate genomic instability. The results illuminate how DNA ends form in non-replicating cells, identify R-loops as the earliest known mutation/amplification intermediate, and suggest that genomic instability during stress could be targeted to transcribed regions, accelerating adaptation.

| S-EPMC3715873 | biostudies-other

Replication fork inhibition in seqA mutants of Escherichia coli triggers replication fork breakage.

Project description:SeqA protein negatively regulates replication initiation in Escherichia coli and is also proposed to organize maturation and segregation of the newly replicated DNA. The seqA mutants suffer from chromosomal fragmentation; since this fragmentation is attributed to defective segregation or nucleoid compaction, two-ended breaks are expected. Instead, we show that, in SeqA's absence, chromosomes mostly suffer one-ended DNA breaks, indicating disintegration of replication forks. We further show that replication forks are unexpectedly slow in seqA mutants. Quantitative kinetics of origin and terminus replication from aligned chromosomes not only confirm origin overinitiation in seqA mutants, but also reveal terminus under-replication, indicating inhibition of replication forks. Pre-/post-labelling studies of the chromosomal fragmentation in seqA mutants suggest events involving single forks, rather than pairs of forks from consecutive rounds rear-ending into each other. We suggest that, in the absence of SeqA, the sister-chromatid cohesion 'safety spacer' is destabilized and completely disappears if the replication fork is inhibited, leading to the segregation fork running into the inhibited replication fork and snapping the latter at single-stranded DNA regions.

| S-EPMC4078979 | biostudies-literature

Chimeras of Escherichia coli and Mycobacterium tuberculosis single-stranded DNA binding proteins: characterization and function in Escherichia coli.

Project description:Single stranded DNA binding proteins (SSBs) are vital for the survival of organisms. Studies on SSBs from the prototype, Escherichia coli (EcoSSB) and, an important human pathogen, Mycobacterium tuberculosis (MtuSSB) had shown that despite significant variations in their quaternary structures, the DNA binding and oligomerization properties of the two are similar. Here, we used the X-ray crystal structure data of the two SSBs to design a series of chimeric proteins (m?1, m?1'?2, m?1-?5, m?1-?6 and m?4-?5) by transplanting ?1, ?1'?2, ?1-?5, ?1-?6 and ?4-?5 regions, respectively of the N-terminal (DNA binding) domain of MtuSSB for the corresponding sequences in EcoSSB. In addition, m?1'?2(ESWR) SSB was generated by mutating the MtuSSB specific 'PRIY' sequence in the ?2 strand of m?1'?2 SSB to EcoSSB specific 'ESWR' sequence. Biochemical characterization revealed that except for m?1 SSB, all chimeras and a control construct lacking the C-terminal domain (?C SSB) bound DNA in modes corresponding to limited and unlimited modes of binding. However, the DNA on MtuSSB may follow a different path than the EcoSSB. Structural probing by protease digestion revealed that unlike other SSBs used, m?1 SSB was also hypersensitive to chymotrypsin treatment. Further, to check for their biological activities, we developed a sensitive assay, and observed that m?1-?6, MtuSSB, m?1'?2 and m?1-?5 SSBs complemented E. coli ?ssb in a dose dependent manner. Complementation by the m?1-?5 SSB was poor. In contrast, m?1'?2(ESWR) SSB complemented E. coli as well as EcoSSB. The inefficiently functioning SSBs resulted in an elongated cell/filamentation phenotype of E. coli. Taken together, our observations suggest that specific interactions within the DNA binding domain of the homotetrameric SSBs are crucial for their biological function.

| S-EPMC3236198 | biostudies-literature

Modulation of Escherichia coli UvrD Single-Stranded DNA Translocation by DNA Base Composition.

Project description:Escherichia coli UvrD is an SF1A DNA helicase/translocase that functions in chromosomal DNA repair and replication of some plasmids. UvrD can also displace proteins such as RecA from DNA in its capacity as an anti-recombinase. Central to all of these activities is its ATP-driven 3'-5' single-stranded (ss) DNA translocation activity. Previous ensemble transient kinetic studies have estimated the average translocation rate of a UvrD monomer on ssDNA composed solely of deoxythymidylates. Here we show that the rate of UvrD monomer translocation along ssDNA is influenced by DNA base composition, with UvrD having the fastest rate along polypyrimidines although decreasing nearly twofold on ssDNA containing equal amounts of the four bases. Experiments with DNA containing abasic sites and polyethylene glycol spacers show that the ssDNA base also influences translocation processivity. These results indicate that changes in base composition and backbone insertions influence the translocation rates, with increased ssDNA base stacking correlated with decreased translocation rates, supporting the proposal that base-stacking interactions are involved in the translocation mechanism.

| S-EPMC5627390 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data