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 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 Artemis-dependent DNA double-strand break formation at stalled replication forks

2013 ◽  
Vol 104 (6) ◽  
pp. 703-710 ◽  
Author(s):  
Junya Unno ◽  
Masatoshi Takagi ◽  
Jinhua Piao ◽  
Masataka Sugimoto ◽  
Fumiko Honda ◽  
...  
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Replication Forks ◽  
Dna Double Strand Break ◽  
Stalled Replication Forks ◽  
Break Formation

scholarly journals Transcription-Associated Recombination Is Dependent on Replication in Mammalian Cells

2007 ◽  
Vol 28 (1) ◽  
pp. 154-164 ◽  
Author(s):  
Ponnari Gottipati ◽  
Tobias N. Cassel ◽  
Linda Savolainen ◽  
Thomas Helleday
Keyword(s):  
Cell Cycle ◽  
Homologous Recombination ◽  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Break ◽  
S Phase ◽  
Replication Forks ◽  
Higher Eukaryotes ◽  
Stalled Replication Forks ◽  
Gene Conversions

ABSTRACT Transcription can enhance recombination; this is a ubiquitous phenomenon from prokaryotes to higher eukaryotes. However, the mechanism of transcription-associated recombination in mammalian cells is poorly understood. Here we have developed a construct with a recombination substrate in which levels of recombination can be studied in the presence or absence of transcription. We observed a direct enhancement in recombination when transcription levels through the substrate were increased. This increase in homologous recombination following transcription is locus specific, since homologous recombination at the unrelated hprt gene is unaffected. In addition, we have shown that transcription-associated recombination involves both short-tract and long-tract gene conversions in mammalian cells, which are different from double-strand-break-induced recombination events caused by endonucleases. Transcription fails to enhance recombination in cells that are not in the S phase of the cell cycle. Furthermore, inhibition of transcription suppresses induction of recombination at stalled replication forks, suggesting that recombination may be involved in bypassing transcription during replication.

scholarly journals The FANCM Ortholog Fml1 Promotes Recombination at Stalled Replication Forks and Limits Crossing Over during DNA Double-Strand Break Repair

2008 ◽  
Vol 32 (1) ◽  
pp. 118-128 ◽  
Author(s):  
Weili Sun ◽  
Saikat Nandi ◽  
Fekret Osman ◽  
Jong Sook Ahn ◽  
Jovana Jakovleska ◽  
...  
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Crossing Over ◽  
Replication Forks ◽  
Dna Double Strand Break ◽  
Break Repair ◽  
Stalled Replication Forks

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 The structure-specific endonuclease complex SLX4/XPF regulates Tus/Ter-induced homologous recombination

2021 ◽  
Author(s):  
Ralph Scully ◽  
Rajula Elango ◽  
Arvind Panday ◽  
Francis Lach ◽  
Nicholas Willis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Replication Fork ◽  
Strand Break ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Site Specific ◽  
A Site ◽  
Break Induced Replication

Abstract Vertebrate replication forks arrested at an interstrand DNA crosslink (ICL) can engage the Fanconi anemia (FA) pathway of ICL repair. The FANCP product, SLX4, binds the FANCQ/XPF/ERCC4-ERCC1 endonuclease, which incises bidirectionally arrested forks to ‘unhook’ the ICL. The resulting double strand break (DSB) is repaired by homologous recombination (HR). Whether this mechanism operates at replication blocks other than ICLs is unknown. Here, we study the role of mammalian SLX4 in HR triggered by a site-specific, chromosomal DNA-protein replication fork barrier formed by the Escherichia coli-derived Tus/Ter complex. We identify an SLX4-XPF-mediated step that is required for Tus/Ter-induced HR but not for HR induced by a replication-independent DSB. We additionally identify a requirement for SLX4-XPF in DSB-induced ‘long tract’ gene conversion, a replicative HR pathway related to break-induced replication. Our work suggests that Tus/Ter-induced HR recapitulates the incision step of replication-coupled ICL repair, and that the full FA mechanism can process DNA-protein barriers for HR.

scholarly journals The RECQL helicase prevents replication fork collapse during replication stress

2020 ◽  
Vol 3 (10) ◽  
pp. e202000668
Author(s):  
Bente Benedict ◽  
Marit AE van Bueren ◽  
Frank PA van Gemert ◽  
Cor Lieftink ◽  
Sergi Guerrero Llobet ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Replication Fork ◽  
Critical Role ◽  
Replication Stress ◽  
Strand Break ◽  
Replication Forks ◽  
Replication Fork Progression ◽  
Dna Dsbs ◽  
Fork Stalling ◽  
Stalled Replication Forks

Most tumors lack the G1/S phase checkpoint and are insensitive to antigrowth signals. Loss of G1/S control can severely perturb DNA replication as revealed by slow replication fork progression and frequent replication fork stalling. Cancer cells may thus rely on specific pathways that mitigate the deleterious consequences of replication stress. To identify vulnerabilities of cells suffering from replication stress, we performed an shRNA-based genetic screen. We report that the RECQL helicase is specifically essential in replication stress conditions and protects stalled replication forks against MRE11-dependent double strand break (DSB) formation. In line with these findings, knockdown of RECQL in different cancer cells increased the level of DNA DSBs. Thus, RECQL plays a critical role in sustaining DNA synthesis under conditions of replication stress and as such may represent a target for cancer therapy.

scholarly journals Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq

PLoS Biology ◽  
2021 ◽  
Vol 19 (3) ◽  
pp. e3000886
Author(s):  
Neesha Kara ◽  
Felix Krueger ◽  
Peter Rugg-Gunn ◽  
Jonathan Houseley
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Replication Fork ◽  
Strand Break ◽  
Double Strand Break ◽  
Replication Forks ◽  
Dna Breaks ◽  
Genome Wide ◽  
Fork Stalling ◽  
End Resection

Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Here, we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3′ ends genome-wide and with base pair resolution. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Replication maps are similar to those obtained by Okazaki fragment sequencing; however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3′ ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

scholarly journals Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq

2020 ◽  
Author(s):  
Neesha Kara ◽  
Felix Krueger ◽  
Peter Rugg-Gunn ◽  
Jonathan Houseley
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Replication Fork ◽  
High Sensitivity ◽  
Strand Break ◽  
Double Strand Break ◽  
Replication Forks ◽  
Dna Breaks ◽  
Biochemical Purification ◽  
Genome Wide

AbstractUnderstanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods sensitive to both changes in replication fork structure and the formation of recombinogenic DNA ends. Here we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single stranded DNA 3’ ends genome-wide and with base pair resolution. TrAEL-seq labels DNA breaks, and profiles both stalled and processive replication forks in yeast and mammalian cells. Replication forks stalling at defined barriers and expressed genes are detected by TrAEL-seq with exceptional signal-to-noise, most likely through labelling of DNA 3’ ends exposed during fork reversal. TrAEL-seq also labels unperturbed processive replication forks to yield maps of replication fork direction similar to those obtained by Okazaki fragment sequencing, however TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3’ ends also allows accurate detection of double strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double strand break hotspots in a dmc1Δ mutant. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

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.

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