scholarly journals Escherichia coli Cells with Increased Levels of DnaA and Deficient in Recombinational Repair Have Decreased Viability

2003 ◽  
Vol 185 (2) ◽  
pp. 630-644 ◽  
Author(s):  
Aline V. Grigorian ◽  
Rachel B. Lustig ◽  
Elena C. Guzmán ◽  
Joseph M. Mahaffy ◽  
Judith W. Zyskind
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Replication Initiation ◽  
Recombinational Repair ◽  
Replication Forks ◽  
Dna Replication Initiation ◽  
Escherichia Coli Cells ◽  
Repair Pathways ◽  
Stalled Replication Forks

ABSTRACT The dnaA operon of Escherichia coli contains the genes dnaA, dnaN, and recF encoding DnaA, β clamp of DNA polymerase III holoenzyme, and RecF. When the DnaA concentration is raised, an increase in the number of DNA replication initiation events but a reduction in replication fork velocity occurs. Because DnaA is autoregulated, these results might be due to the inhibition of dnaN and recF expression. To test this, we examined the effects of increasing the intracellular concentrations of DnaA, β clamp, and RecF, together and separately, on initiation, the rate of fork movement, and cell viability. The increased expression of one or more of the dnaA operon proteins had detrimental effects on the cell, except in the case of RecF expression. A shorter C period was not observed with increased expression of the β clamp; in fact, many chromosomes did not complete replication in runout experiments. Increased expression of DnaA alone resulted in stalled replication forks, filamentation, and a decrease in viability. When the three proteins of the dnaA operon were simultaneously overexpressed, highly filamentous cells were observed (>50 μm) with extremely low viability and, in runout experiments, most chromosomes had not completed replication. The possibility that recombinational repair was responsible for the survival of cells overexpressing DnaA was tested by using mutants in different recombinational repair pathways. The absence of RecA, RecB, RecC, or the proteins in the RuvABC complex caused an additional ∼100-fold drop in viability in cells with increased levels of DnaA, indicating a requirement for recombinational repair in these cells.

scholarly journals Double-Strand Break Generation under Deoxyribonucleotide Starvation in Escherichia coli

2007 ◽  
Vol 189 (15) ◽  
pp. 5782-5786 ◽  
Author(s):  
Estrella Guarino ◽  
Israel Salguero ◽  
Alfonso Jiménez-Sánchez ◽  
Elena C. Guzmán
Keyword(s):  
Escherichia Coli ◽  
Replication Fork ◽  
Thermal Inactivation ◽  
Strand Break ◽  
Double Strand Break ◽  
Replication Forks ◽  
Thymine Starvation ◽  
Hydroxyurea Treatment ◽  
Deoxynucleoside Triphosphate ◽  
Stalled Replication Forks

ABSTRACT Stalled replication forks produced by three different ways of depleting deoxynucleoside triphosphate showed different capacities to undergo “replication fork reversal.” This reaction occurred at the stalled forks generated by hydroxyurea treatment, was impaired under thermal inactivation of ribonucleoside reductase, and did not take place under thymine starvation.

scholarly journals Phenotypes of dnaXE145A Mutant Cells Indicate that the Escherichia coli Clamp Loader Has a Role in the Restart of Stalled Replication Forks

2017 ◽  
Vol 199 (24) ◽  
Author(s):  
Ingvild Flåtten ◽  
Emily Helgesen ◽  
Ida Benedikte Pedersen ◽  
Torsten Waldminghaus ◽  
Christiane Rothe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Replication Fork ◽  
Temperature Sensitive ◽  
Replication Forks ◽  
Clamp Loader ◽  
Topoisomerase Iv ◽  
Content Type ◽  
Stalled Replication Forks ◽  
Mutant Cells

ABSTRACT The Escherichia coli dnaXE145A mutation was discovered in connection with a screen for multicopy suppressors of the temperature-sensitive topoisomerase IV mutation parE10. The gene for the clamp loader subunits τ and γ, dnaX, but not the mutant dnaXE145A , was found to suppress parE10(Ts) when overexpressed. Purified mutant protein was found to be functional in vitro, and few phenotypes were found in vivo apart from problems with partitioning of DNA in rich medium. We show here that a large number of the replication forks that initiate at oriC never reach the terminus in dnaXE145A mutant cells. The SOS response was found to be induced, and a combination of the dnaXE145A mutation with recBC and recA mutations led to reduced viability. The mutant cells exhibited extensive chromosome fragmentation and degradation upon inactivation of recBC and recA, respectively. The results indicate that the dnaXE145A mutant cells suffer from broken replication forks and that these need to be repaired by homologous recombination. We suggest that the dnaX-encoded τ and γ subunits of the clamp loader, or the clamp loader complex itself, has a role in the restart of stalled replication forks without extensive homologous recombination. IMPORTANCE The E. coli clamp loader complex has a role in coordinating the activity of the replisome at the replication fork and loading β-clamps for lagging-strand synthesis. Replication forks frequently encounter obstacles, such as template lesions, secondary structures, and tightly bound protein complexes, which will lead to fork stalling. Some pathways of fork restart have been characterized, but much is still unknown about the actors and mechanisms involved. We have in this work characterized the dnaXE145A clamp loader mutant. We find that the naturally occurring obstacles encountered by a replication fork are not tackled in a proper way by the mutant clamp loader and suggest a role for the clamp loader in the restart of stalled replication forks.

scholarly journals Mechanisms of replication fork restart in Escherichia coli

2004 ◽  
Vol 359 (1441) ◽  
pp. 71-77 ◽  
Author(s):  
Kenneth J. Marians
Keyword(s):  
Escherichia Coli ◽  
High Frequency ◽  
Genetic Information ◽  
Replication Fork ◽  
Replication Forks ◽  
Replication Proteins ◽  
Replication Restart ◽  
Stalled Replication Forks

Replication of the genome is crucial for the accurate transmission of genetic information. It has become clear over the last decade that the orderly progression of replication forks in both prokaryotes and eukaryotes is disrupted with high frequency by encounters with various obstacles either on or in the template strands. Survival of the organism then becomes dependent on both removal of the obstruction and resumption of replication. This latter point is particularly important in bacteria, where the number of replication forks per genome is nominally only two. Replication restart in Escherichia coli is accomplished by the action of the restart primosomal proteins, which use both recombination intermediates and stalled replication forks as substrates for loading new replication forks. These reactions have been reconstituted with purified recombination and replication proteins.

scholarly journals Destabilization of the holo-DNA Polδ by loss of Pol32 confers conditional lethality that can be suppressed by stabilizing Pol31-Pol3 interaction

2021 ◽  
Author(s):  
Kenji Shimada ◽  
Monika Tsai-Pflugfelder ◽  
Niloofar Davoodi Vijeh Motlagh ◽  
Neda Delgoshaie ◽  
Jeannette Fuchs ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Replication Fork ◽  
Essential Role ◽  
Genome Integrity ◽  
Genome Replication ◽  
Replication Forks ◽  
Kinase Activation ◽  
Yeast Strains ◽  
Stalled Replication Forks ◽  
Conserved Amino Acids

AbstractDNA Polymerase δ plays an essential role in genome replication and in the preservation of genome integrity. In S. cerevisiae, Polδ consists of three subunits: Pol3 (the catalytic subunit), Pol31 and Pol32. We have constructed pol31 mutants by alanine substitution at conserved amino acids, and identified three alleles that do not confer any disadvantage on their own, but which suppress the cold-, HU- and MMS-hypersensitivity of yeast strains lacking Pol32. We have shown that Pol31 and Pol32 are both involved in translesion synthesis, error-free bypass synthesis, and in preservation of replication fork stability under conditions of HU arrest. We identified a solvent exposed loop in Pol31 defined by two alanine substitutions at T415 and W417. Whereas pol31-T4l5A compromises polymerase stability at stalled forks, pol31-W417A is able to suppress many, but not all, of the phenotypes arising from pol32Δ. ChIP analyses showed that the absence of Pol32 destabilizes Pole and Polα at stalled replication forks, but does not interfere with checkpoint kinase activation. We show that the Pol31-W417A-mediated suppression of replicationstress sensitivity in pol32Δ stems from enhanced interaction between Pol3 and Pol31, which stabilizes a functional Polδ.

scholarly journals Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

2015 ◽  
Vol 197 (17) ◽  
pp. 2792-2809 ◽  
Author(s):  
Sarita Mallik ◽  
Ellen M. Popodi ◽  
Andrew J. Hanson ◽  
Patricia L. Foster
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Dna Helicase ◽  
Dna Lesions ◽  
Replication Forks ◽  
Content Type ◽  
Pol Iv ◽  
Dna Polymerase Iv ◽  
Stalled Replication Forks

ABSTRACTEscherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure ofE. colito DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that thein vitrointeraction between Rep and Pol IV reported previously also occursin vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecAin vivoand is recruited to sites of DSBs to aid in the restoration of DNA replication.IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstratein vivolocalization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings providein vivoevidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

scholarly journals Delineation of the Ancestral Tus-Dependent Replication Fork Trap

2021 ◽  
Author(s):  
Casey Toft ◽  
Morgane Moreau ◽  
Jiri Perutka ◽  
Savitri Mandapati ◽  
Peter Enyeart ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Replication Fork ◽  
Wide Distribution ◽  
Replication Forks ◽  
Genome Wide ◽  
Replication Fork Arrest

In Escherichia coli, DNA replication termination is orchestrated by two clusters of Ter sites forming a DNA replication fork trap when bound by Tus proteins. The formation of a ‘locked’ Tus-Ter complex is essential for halting incoming DNA replication forks. However, the absence of replication fork arrest at some Ter sites raised questions about their significance. In this study, we examined the genome-wide distribution of Tus and found that only the six innermost Ter sites (TerA-E and G) were significantly bound by Tus. We also found that a single ectopic insertion of TerB in its non-permissive orientation could not be achieved, advocating against a need for ‘back-up’ Ter sites. Finally, examination of the genomes of a variety of Enterobacterales revealed a new replication fork trap architecture mostly found outside the Enterobacteriaceae family. Taken together, our data enabled the delineation of a narrow ancestral Tus-dependent DNA replication fork trap consisting of only two Ter sites.

scholarly journals Replication fork reversal in DNA polymerase III mutants of Escherichia coli: a role for the β clamp

2002 ◽  
Vol 44 (5) ◽  
pp. 1331-1339 ◽  
Author(s):  
Gianfranco Grompone ◽  
Marie Seigneur ◽  
S. Dusko Ehrlich ◽  
Bénédicte Michel
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Dna Polymerase Iii ◽  
Polymerase Iii
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