13 results on '"Jergic, Slobodan"'
Search Results
2. Single-molecule visualization of stalled replication-fork rescue by the Escherichia coli Rep helicase.
- Author
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Whinn KS, Xu ZQ, Jergic S, Sharma N, Spenkelink LM, Dixon NE, van Oijen AM, and Ghodke H
- Subjects
- DNA metabolism, DNA Replication, Escherichia coli enzymology, DNA Helicases chemistry, Escherichia coli Proteins chemistry
- Abstract
Genome duplication occurs while the template DNA is bound by numerous DNA-binding proteins. Each of these proteins act as potential roadblocks to the replication fork and can have deleterious effects on cells. In Escherichia coli, these roadblocks are displaced by the accessory helicase Rep, a DNA translocase and helicase that interacts with the replisome. The mechanistic details underlying the coordination with replication and roadblock removal by Rep remain poorly understood. Through real-time fluorescence imaging of the DNA produced by individual E. coli replisomes and the simultaneous visualization of fluorescently-labeled Rep, we show that Rep continually surveils elongating replisomes. We found that this association of Rep with the replisome is stochastic and occurs independently of whether the fork is stalled or not. Further, we visualize the efficient rescue of stalled replication forks by directly imaging individual Rep molecules as they remove a model protein roadblock, dCas9, from the template DNA. Using roadblocks of varying DNA-binding stabilities, we conclude that continuation of synthesis is the rate-limiting step of stalled replication rescue., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2023
- Full Text
- View/download PDF
3. Mechanism of transcription modulation by the transcription-repair coupling factor.
- Author
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Paudel BP, Xu ZQ, Jergic S, Oakley AJ, Sharma N, Brown SHJ, Bouwer JC, Lewis PJ, Dixon NE, van Oijen AM, and Ghodke H
- Subjects
- DNA-Directed RNA Polymerases genetics, DNA-Directed RNA Polymerases metabolism, Transcription, Genetic, Bacterial Proteins genetics, Bacterial Proteins metabolism, DNA Repair, Escherichia coli genetics, Escherichia coli metabolism, Transcription Factors genetics, Transcription Factors metabolism
- Abstract
Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes paused/stalled RNAPs and either rescues transcription or initiates transcription termination. Precisely how TRCFs choose to execute either outcome remains unclear. With Escherichia coli as a model, we used single-molecule assays to study dynamic modulation of elongation by Mfd, the bacterial TRCF. We found that nucleotide-bound Mfd converts the elongation complex (EC) into a catalytically poised state, presenting the EC with an opportunity to restart transcription. After long-lived residence in this catalytically poised state, ATP hydrolysis by Mfd remodels the EC through an irreversible process leading to loss of the RNA transcript. Further, biophysical studies revealed that the motor domain of Mfd binds and partially melts DNA containing a template strand overhang. The results explain pathway choice determining the fate of the EC and provide a molecular mechanism for transcription modulation by TRCF., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2022
- Full Text
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4. DnaB helicase dynamics in bacterial DNA replication resolved by single-molecule studies.
- Author
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Spinks RR, Spenkelink LM, Stratmann SA, Xu ZQ, Stamford NPJ, Brown SE, Dixon NE, Jergic S, and van Oijen AM
- Subjects
- DNA-Directed DNA Polymerase, Escherichia coli genetics, Multienzyme Complexes, Single Molecule Imaging, DNA Replication, DnaB Helicases metabolism
- Abstract
In Escherichia coli, the DnaB helicase forms the basis for the assembly of the DNA replication complex. The stability of DnaB at the replication fork is likely important for successful replication initiation and progression. Single-molecule experiments have significantly changed the classical model of highly stable replication machines by showing that components exchange with free molecules from the environment. However, due to technical limitations, accurate assessments of DnaB stability in the context of replication are lacking. Using in vitro fluorescence single-molecule imaging, we visualise DnaB loaded on forked DNA templates. That these helicases are highly stable at replication forks, indicated by their observed dwell time of ∼30 min. Addition of the remaining replication factors results in a single DnaB helicase integrated as part of an active replisome. In contrast to the dynamic behaviour of other replisome components, DnaB is maintained within the replisome for the entirety of the replication process. Interestingly, we observe a transient interaction of additional helicases with the replication fork. This interaction is dependent on the τ subunit of the clamp-loader complex. Collectively, our single-molecule observations solidify the role of the DnaB helicase as the stable anchor of the replisome, but also reveal its capacity for dynamic interactions., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2021
- Full Text
- View/download PDF
5. Single-molecule live-cell imaging reveals RecB-dependent function of DNA polymerase IV in double strand break repair.
- Author
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Henrikus SS, Henry C, McGrath AE, Jergic S, McDonald JP, Hellmich Y, Bruckbauer ST, Ritger ML, Cherry ME, Wood EA, Pham PT, Goodman MF, Woodgate R, Cox MM, van Oijen AM, Ghodke H, and Robinson A
- Subjects
- Ciprofloxacin pharmacology, DNA Damage drug effects, DNA Polymerase beta genetics, DNA Repair genetics, DNA Replication genetics, Escherichia coli genetics, Escherichia coli ultrastructure, Exodeoxyribonuclease V genetics, Single Molecule Imaging, DNA Breaks, Double-Stranded drug effects, DNA Polymerase beta ultrastructure, DNA-Binding Proteins genetics, Escherichia coli Proteins genetics, Escherichia coli Proteins ultrastructure, Exodeoxyribonuclease V ultrastructure, Rec A Recombinases genetics
- Abstract
Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2020
- Full Text
- View/download PDF
6. Development of a single-stranded DNA-binding protein fluorescent fusion toolbox.
- Author
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Dubiel K, Henry C, Spenkelink LM, Kozlov AG, Wood EA, Jergic S, Dixon NE, van Oijen AM, Cox MM, Lohman TM, Sandler SJ, and Keck JL
- Subjects
- DNA Damage, DNA Repair, DNA Replication, DNA, Single-Stranded chemistry, Escherichia coli cytology, Escherichia coli genetics, Escherichia coli metabolism, Genome, Bacterial, Intrinsically Disordered Proteins chemistry, Protein Binding, SOS Response, Genetics, DNA-Binding Proteins analysis, DNA-Binding Proteins chemistry, Fluorescence, Recombinant Fusion Proteins analysis, Recombinant Fusion Proteins chemistry
- Abstract
Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein attached directly to the C-terminus that is similar to fusions used in previous studies displayed dysfunctional protein interaction activity. The SSB-IDL fusions are readily visualized in single-molecule DNA replication reactions. Escherichia coli strains in which wildtype SSB is replaced by SSB-IDL fusions are viable and display normal growth rates and fitness. The SSB-IDL fusions form detectible SSB foci in cells with frequencies mirroring previously examined fluorescent DNA replication fusion proteins. Cells expressing SSB-IDL fusions are sensitized to some DNA damaging agents. The results highlight the utility of SSB-IDL fusions for biochemical and cellular studies of genome maintenance reactions., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2020
- Full Text
- View/download PDF
7. Recycling of single-stranded DNA-binding protein by the bacterial replisome.
- Author
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Spenkelink LM, Lewis JS, Jergic S, Xu ZQ, Robinson A, Dixon NE, and van Oijen AM
- Subjects
- DNA genetics, DNA metabolism, DNA Polymerase III genetics, DNA Polymerase III metabolism, DNA Primase genetics, DNA Primase metabolism, DNA, Bacterial metabolism, DNA, Single-Stranded chemistry, DNA, Single-Stranded metabolism, DnaB Helicases genetics, DnaB Helicases metabolism, Escherichia coli metabolism, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Time-Lapse Imaging, DNA Replication, DNA, Bacterial genetics, DNA, Single-Stranded genetics, Escherichia coli genetics
- Abstract
Single-stranded DNA-binding proteins (SSBs) support DNA replication by protecting single-stranded DNA from nucleolytic attack, preventing intra-strand pairing events and playing many other regulatory roles within the replisome. Recent developments in single-molecule approaches have led to a revised picture of the replisome that is much more complex in how it retains or recycles protein components. Here, we visualize how an in vitro reconstituted Escherichia coli replisome recruits SSB by relying on two different molecular mechanisms. Not only does it recruit new SSB molecules from solution to coat newly formed single-stranded DNA on the lagging strand, but it also internally recycles SSB from one Okazaki fragment to the next. We show that this internal transfer mechanism is balanced against recruitment from solution in a manner that is concentration dependent. By visualizing SSB dynamics in live cells, we show that both internal transfer and external exchange mechanisms are physiologically relevant., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2019
- Full Text
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8. Exchange between Escherichia coli polymerases II and III on a processivity clamp.
- Author
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Kath JE, Chang S, Scotland MK, Wilbertz JH, Jergic S, Dixon NE, Sutton MD, and Loparo JJ
- Subjects
- DNA genetics, DNA Damage genetics, DNA Polymerase II chemistry, DNA Polymerase III chemistry, DNA Polymerase beta chemistry, DNA Repair genetics, DNA Replication genetics, Escherichia coli enzymology, Escherichia coli genetics, Multiprotein Complexes chemistry, Multiprotein Complexes genetics, Protein Structure, Tertiary, DNA biosynthesis, DNA Polymerase II genetics, DNA Polymerase III genetics, DNA Polymerase beta genetics
- Abstract
Escherichia coli has three DNA polymerases implicated in the bypass of DNA damage, a process called translesion synthesis (TLS) that alleviates replication stalling. Although these polymerases are specialized for different DNA lesions, it is unclear if they interact differently with the replication machinery. Of the three, DNA polymerase (Pol) II remains the most enigmatic. Here we report a stable ternary complex of Pol II, the replicative polymerase Pol III core complex and the dimeric processivity clamp, β. Single-molecule experiments reveal that the interactions of Pol II and Pol III with β allow for rapid exchange during DNA synthesis. As with another TLS polymerase, Pol IV, increasing concentrations of Pol II displace the Pol III core during DNA synthesis in a minimal reconstitution of primer extension. However, in contrast to Pol IV, Pol II is inefficient at disrupting rolling-circle synthesis by the fully reconstituted Pol III replisome. Together, these data suggest a β-mediated mechanism of exchange between Pol II and Pol III that occurs outside the replication fork., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2016
- Full Text
- View/download PDF
9. Two mechanisms coordinate replication termination by the Escherichia coli Tus-Ter complex.
- Author
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Pandey M, Elshenawy MM, Jergic S, Takahashi M, Dixon NE, Hamdan SM, and Patel SS
- Subjects
- Base Pairing, DNA biosynthesis, DNA chemistry, DNA Helicases metabolism, DNA-Directed DNA Polymerase metabolism, Models, Genetic, DNA Replication, DNA-Binding Proteins metabolism, Escherichia coli Proteins metabolism
- Abstract
The Escherichia coli replication terminator protein (Tus) binds to Ter sequences to block replication forks approaching from one direction. Here, we used single molecule and transient state kinetics to study responses of the heterologous phage T7 replisome to the Tus-Ter complex. The T7 replisome was arrested at the non-permissive end of Tus-Ter in a manner that is explained by a composite mousetrap and dynamic clamp model. An unpaired C(6) that forms a lock by binding into the cytosine binding pocket of Tus was most effective in arresting the replisome and mutation of C(6) removed the barrier. Isolated helicase was also blocked at the non-permissive end, but unexpectedly the isolated polymerase was not, unless C(6) was unpaired. Instead, the polymerase was blocked at the permissive end. This indicates that the Tus-Ter mechanism is sensitive to the translocation polarity of the DNA motor. The polymerase tracking along the template strand traps the C(6) to prevent lock formation; the helicase tracking along the other strand traps the complementary G(6) to aid lock formation. Our results are consistent with the model where strand separation by the helicase unpairs the GC(6) base pair and triggers lock formation immediately before the polymerase can sequester the C(6) base., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2015
- Full Text
- View/download PDF
10. Proofreading exonuclease on a tether: the complex between the E. coli DNA polymerase III subunits α, epsilon, θ and β reveals a highly flexible arrangement of the proofreading domain.
- Author
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Ozawa K, Horan NP, Robinson A, Yagi H, Hill FR, Jergic S, Xu ZQ, Loscha KV, Li N, Tehei M, Oakley AJ, Otting G, Huber T, and Dixon NE
- Subjects
- Amino Acid Sequence, DNA Polymerase III metabolism, Escherichia coli Proteins metabolism, Exodeoxyribonucleases metabolism, Models, Molecular, Molecular Sequence Data, Peptides chemistry, Protein Folding, Protein Interaction Domains and Motifs, Protein Structure, Tertiary, DNA Polymerase III chemistry, Escherichia coli Proteins chemistry, Exodeoxyribonucleases chemistry
- Abstract
A complex of the three (αεθ) core subunits and the β2 sliding clamp is responsible for DNA synthesis by Pol III, the Escherichia coli chromosomal DNA replicase. The 1.7 Å crystal structure of a complex between the PHP domain of α (polymerase) and the C-terminal segment of ε (proofreading exonuclease) subunits shows that ε is attached to α at a site far from the polymerase active site. Both α and ε contain clamp-binding motifs (CBMs) that interact simultaneously with β2 in the polymerization mode of DNA replication by Pol III. Strengthening of both CBMs enables isolation of stable αεθ:β2 complexes. Nuclear magnetic resonance experiments with reconstituted αεθ:β2 demonstrate retention of high mobility of a segment of 22 residues in the linker that connects the exonuclease domain of ε with its α-binding segment. In spite of this, small-angle X-ray scattering data show that the isolated complex with strengthened CBMs has a compact, but still flexible, structure. Photo-crosslinking with p-benzoyl-L-phenylalanine incorporated at different sites in the α-PHP domain confirm the conformational variability of the tether. Structural models of the αεθ:β2 replicase complex with primer-template DNA combine all available structural data.
- Published
- 2013
- Full Text
- View/download PDF
11. The proofreading exonuclease subunit epsilon of Escherichia coli DNA polymerase III is tethered to the polymerase subunit alpha via a flexible linker.
- Author
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Ozawa K, Jergic S, Park AY, Dixon NE, and Otting G
- Subjects
- Amino Acid Sequence, Cell-Free System, DNA-Directed DNA Polymerase chemistry, Molecular Sequence Data, Nitrogen Isotopes, Nuclear Magnetic Resonance, Biomolecular, Protein Structure, Tertiary, Sequence Homology, Amino Acid, DNA Polymerase III chemistry, Escherichia coli Proteins chemistry, Exodeoxyribonucleases chemistry
- Abstract
Escherichia coli DNA polymerase III holoenzyme is composed of 10 different subunits linked by noncovalent interactions. The polymerase activity resides in the alpha-subunit. The epsilon-subunit, which contains the proofreading exonuclease site within its N-terminal 185 residues, binds to alpha via a segment of 57 additional C-terminal residues, and also to theta, whose function is less well defined. The present study shows that theta greatly enhances the solubility of epsilon during cell-free synthesis. In addition, synthesis of epsilon in the presence of theta and alpha resulted in a soluble ternary complex that could readily be purified and analyzed by NMR spectroscopy. Cell-free synthesis of epsilon from PCR-amplified DNA coupled with site-directed mutagenesis and selective 15N-labeling provided site-specific assignments of NMR resonances of epsilon that were confirmed by lanthanide-induced pseudocontact shifts. The data show that the proofreading domain of epsilon is connected to alpha via a flexible linker peptide comprising over 20 residues. This distinguishes the alpha : epsilon complex from other proofreading polymerases, which have a more rigid multidomain structure.
- Published
- 2008
- Full Text
- View/download PDF
12. Solution structure of Domains IVa and V of the tau subunit of Escherichia coli DNA polymerase III and interaction with the alpha subunit.
- Author
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Su XC, Jergic S, Keniry MA, Dixon NE, and Otting G
- Subjects
- Base Sequence, Binding Sites, DNA Polymerase III metabolism, Escherichia coli Proteins metabolism, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Protein Folding, Protein Structure, Tertiary, Protein Subunits chemistry, Protein Subunits metabolism, Sequence Alignment, Solutions, Transcription Factors metabolism, DNA Polymerase III chemistry, Escherichia coli Proteins chemistry, Models, Molecular, Transcription Factors chemistry
- Abstract
The solution structure of the C-terminal Domain V of the tau subunit of E. coli DNA polymerase III was determined by nuclear magnetic resonance (NMR) spectroscopy. The fold is unique to tau subunits. Amino acid sequence conservation is pronounced for hydrophobic residues that form the structural core of the protein, indicating that the fold is representative for tau subunits from a wide range of different bacteria. The interaction between the polymerase subunits tau and alpha was studied by NMR experiments where alpha was incubated with full-length C-terminal domain (tau(C)16), and domains shortened at the C-terminus by 11 and 18 residues, respectively. The only interacting residues were found in the C-terminal 30-residue segment of tau, most of which is structurally disordered in free tau(C)16. Since the N- and C-termini of the structured core of tau(C)16 are located close to each other, this limits the possible distance between alpha and the pentameric deltatau2gammadelta' clamp-loader complex and, hence, between the two alpha subunits involved in leading- and lagging-strand DNA synthesis. Analysis of an N-terminally extended construct (tau(C)22) showed that tau(C)14 presents the only part of Domains IVa and V of tau which comprises a globular fold in the absence of other interaction partners.
- Published
- 2007
- Full Text
- View/download PDF
13. The unstructured C-terminus of the tau subunit of Escherichia coli DNA polymerase III holoenzyme is the site of interaction with the alpha subunit.
- Author
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Jergic S, Ozawa K, Williams NK, Su XC, Scott DD, Hamdan SM, Crowther JA, Otting G, and Dixon NE
- Subjects
- Binding Sites, DNA metabolism, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Mutagenesis, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, Protein Subunits chemistry, Protein Subunits genetics, Protein Subunits metabolism, Surface Plasmon Resonance, Transcription Factors genetics, Transcription Factors metabolism, DNA Polymerase III metabolism, Escherichia coli Proteins chemistry, Transcription Factors chemistry
- Abstract
The tau subunit of Escherichia coli DNA polymerase III holoenzyme interacts with the alpha subunit through its C-terminal Domain V, tau(C)16. We show that the extreme C-terminal region of tau(C)16 constitutes the site of interaction with alpha. The tau(C)16 domain, but not a derivative of it with a C-terminal deletion of seven residues (tau(C)16Delta7), forms an isolable complex with alpha. Surface plasmon resonance measurements were used to determine the dissociation constant (K(D)) of the alpha-tau(C)16 complex to be approximately 260 pM. Competition with immobilized tau(C)16 by tau(C)16 derivatives for binding to alpha gave values of K(D) of 7 muM for the alpha-tau(C)16Delta7 complex. Low-level expression of the genes encoding tau(C)16 and tau(C)16triangle up7, but not tau(C)16Delta11, is lethal to E. coli. Suppression of this lethal phenotype enabled selection of mutations in the 3' end of the tau(C)16 gene, that led to defects in alpha binding. The data suggest that the unstructured C-terminus of tau becomes folded into a helix-loop-helix in its complex with alpha. An N-terminally extended construct, tau(C)24, was found to bind DNA in a salt-sensitive manner while no binding was observed for tau(C)16, suggesting that the processivity switch of the replisome functionally involves Domain IV of tau.
- Published
- 2007
- Full Text
- View/download PDF
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