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1.
Elife ; 122024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38315099

RESUMO

Structural maintenance of chromosomes (SMC) complexes share conserved structures and serve a common role in maintaining chromosome architecture. In the bacterium Escherichia coli, the SMC complex MukBEF is necessary for rapid growth and the accurate segregation and positioning of the chromosome, although the specific molecular mechanisms involved are still unknown. Here, we used a number of in vivo assays to reveal how MukBEF controls chromosome conformation and how the MatP/matS system prevents MukBEF activity. Our results indicate that the loading of MukBEF occurs preferentially on newly replicated DNA, at multiple loci on the chromosome where it can promote long-range contacts in cis even though MukBEF can promote long-range contacts in the absence of replication. Using Hi-C and ChIP-seq analyses in strains with rearranged chromosomes, the prevention of MukBEF activity increases with the number of matS sites and this effect likely results from the unloading of MukBEF by MatP. Altogether, our results reveal how MukBEF operates to control chromosome folding and segregation in E. coli.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas Repressoras/genética , Cromossomos Bacterianos/genética , Origem de Replicação , Proteínas Cromossômicas não Histona/genética , Cromossomos , Segregação de Cromossomos
2.
Cell ; 172(4): 771-783.e18, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29358050

RESUMO

As in eukaryotes, bacterial genomes are not randomly folded. Bacterial genetic information is generally carried on a circular chromosome with a single origin of replication from which two replication forks proceed bidirectionally toward the opposite terminus region. Here, we investigate the higher-order architecture of the Escherichia coli genome, showing its partition into two structurally distinct entities by a complex and intertwined network of contacts: the replication terminus (ter) region and the rest of the chromosome. Outside of ter, the condensin MukBEF and the ubiquitous nucleoid-associated protein (NAP) HU promote DNA contacts in the megabase range. Within ter, the MatP protein prevents MukBEF activity, and contacts are restricted to ∼280 kb, creating a domain with distinct structural properties. We also show how other NAPs contribute to nucleoid organization, such as H-NS, which restricts short-range interactions. Combined, these results reveal the contributions of major evolutionarily conserved proteins in a bacterial chromosome organization.


Assuntos
Adenosina Trifosfatases , Cromossomos Bacterianos , Proteínas de Ligação a DNA , Escherichia coli K12 , Complexos Multiproteicos , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , Cromossomos Bacterianos/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Escherichia coli K12/ultraestrutura , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Estrutura Quaternária de Proteína , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
3.
PLoS Genet ; 13(5): e1006758, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28486476

RESUMO

The Escherichia coli chromosome is organized into four macrodomains (Ori, Ter, Right and Left) and two non-structured regions. This organization influences the segregation of sister chromatids, the mobility of chromosomal DNA, and the cellular localization of the chromosome. The organization of the Ter and Ori macrodomains relies on two specific systems, MatP/matS for the Ter domain and MaoP/maoS for the Ori domain, respectively. Here by constructing strains with chromosome rearrangements to reshuffle the distribution of chromosomal segments, we reveal that the difference between the non-structured regions and the Right and Left lateral macrodomains relies on their position on the chromosome. A change in the genetic location of oriC generated either by an inversion within the Ori macrodomain or by the insertion of a second oriC modifies the position of Right and Left macrodomains, as the chromosome region the closest to oriC are always non-structured while the regions further away behave as macrodomain regardless of their DNA sequence. Using fluorescent microscopy we estimated that loci belonging to a non-structured region are significantly closer to the Ori MD than loci belonging to a lateral MD. Altogether, our results suggest that the origin of replication plays a prominent role in chromosome organization in E. coli, as it determines structuring and localization of macrodomains in growing cell.


Assuntos
Cromossomos Bacterianos/genética , Escherichia coli/genética , Origem de Replicação , Mapeamento Cromossômico , Proteínas de Escherichia coli/genética , Loci Gênicos
4.
PLoS Genet ; 10(10): e1004719, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25329071

RESUMO

The HolC-HolD (χψ) complex is part of the DNA polymerase III holoenzyme (Pol III HE) clamp-loader. Several lines of evidence indicate that both leading- and lagging-strand synthesis are affected in the absence of this complex. The Escherichia coli ΔholD mutant grows poorly and suppressor mutations that restore growth appear spontaneously. Here we show that duplication of the ssb gene, encoding the single-stranded DNA binding protein (SSB), restores ΔholD mutant growth at all temperatures on both minimal and rich medium. RecFOR-dependent SOS induction, previously shown to occur in the ΔholD mutant, is unaffected by ssb gene duplication, suggesting that lagging-strand synthesis remains perturbed. The C-terminal SSB disordered tail, which interacts with several E. coli repair, recombination and replication proteins, must be intact in both copies of the gene in order to restore normal growth. This suggests that SSB-mediated ΔholD suppression involves interaction with one or more partner proteins. ssb gene duplication also suppresses ΔholC single mutant and ΔholC ΔholD double mutant growth defects, indicating that it bypasses the need for the entire χψ complex. We propose that doubling the amount of SSB stabilizes HolCD-less Pol III HE DNA binding through interactions between SSB and a replisome component, possibly DnaE. Given that SSB binds DNA in vitro via different binding modes depending on experimental conditions, including SSB protein concentration and SSB interactions with partner proteins, our results support the idea that controlling the balance between SSB binding modes is critical for DNA Pol III HE stability in vivo, with important implications for DNA replication and genome stability.


Assuntos
DNA Polimerase III/genética , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , DNA Polimerase III/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Duplicação Gênica , Regulação Bacteriana da Expressão Gênica , Mutação , Resposta SOS em Genética , Supressão Genética , Temperatura
5.
PLoS Genet ; 8(4): e1002622, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22496668

RESUMO

Replication fork arrest is a recognized source of genetic instability, and transcription is one of the most prominent causes of replication impediment. We analyze here the requirement for recombination proteins in Escherichia coli when replication-transcription head-on collisions are induced at a specific site by the inversion of a highly expressed ribosomal operon (rrn). RecBC is the only recombination protein required for cell viability under these conditions of increased replication-transcription collisions. In its absence, fork breakage occurs at the site of collision, and the resulting linear DNA is not repaired and is slowly degraded by the RecJ exonuclease. Lethal fork breakage is also observed in cells that lack RecA and RecD, i.e. when both homologous recombination and the potent exonuclease V activity of the RecBCD complex are inactivated, with a slow degradation of the resulting linear DNA by the combined action of the RecBC helicase and the RecJ exonuclease. The sizes of the major linear fragments indicate that DNA degradation is slowed down by the encounter with another rrn operon. The amount of linear DNA decreases nearly two-fold when the Holliday junction resolvase RuvABC is inactivated in recB, as well as in recA recD mutants, indicating that part of the linear DNA is formed by resolution of a Holliday junction. Our results suggest that replication fork reversal occurs after replication-transcription head-on collision, and we propose that it promotes the action of the accessory replicative helicases that dislodge the obstacle.


Assuntos
Replicação do DNA/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Exodesoxirribonuclease V/genética , Recombinação Homóloga , Metiltransferases , Proteínas de Bactérias/genética , Fragmentação do DNA , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Cruciforme/metabolismo , Exodesoxirribonucleases/genética , Resolvases de Junção Holliday/genética , Recombinação Homóloga/genética , Metiltransferases/genética , Metiltransferases/metabolismo , Mutação , Recombinases Rec A/genética , Transcrição Gênica
6.
Mol Microbiol ; 77(2): 324-36, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20497334

RESUMO

We observed that cells lacking Rep and UvrD, two replication accessory helicases, and the recombination protein RecF are cryo-sensitive on rich medium. We isolated five mutations that suppress this Luria-Bertani (LB)-cryo-sensitivity and show that they map in the genes encoding the RNA polymerase subunits RpoB and RpoC. These rpoB (D444G, H447R and N518D) and rpoC mutants (H113R and P451L) were characterized. rpoB(H447R) and rpoB(D444G) prevent activation of the Prrn core promoter in rich medium, but only rpoB(H447R) also suppresses the auxotrophy of a relA spoT mutant (stringent-like phenotype). rpoC(H113R) suppresses the thermo-sensitivity of a greA greB mutant, suggesting that it destabilizes stalled elongation complexes. All mutations but rpoC(P451L) prevent R-loop formation. We propose that these rpo mutations allow replication in the absence of Rep and UvrD by destabilizing RNA Pol upon replication-transcription collisions. In a RecF(+) context, they improve growth of rep uvrD cells only if DinG is present, supporting the hypothesis that Rep, UvrD and DinG facilitate progression of the replication fork across transcribed sequences. They rescue rep uvrD dinG recF cells, indicating that in a recF mutant replication forks arrested by unstable transcription complexes can restart without any of the three known replication accessory helicases Rep, UvrD and DinG.


Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Temperatura Baixa , DNA Helicases/genética , Replicação do DNA , DNA Bacteriano/biossíntese , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Supressão Genética , Raios Ultravioleta
7.
PLoS Pathog ; 5(11): e1000663, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19936046

RESUMO

The Vibrionaceae is comprised of numerous aquatic species and includes several human pathogens, such as Vibrio cholerae, the cause of cholera. All organisms in this family have two chromosomes, and replication of the smaller one depends on rctB, a gene that is restricted to the Vibrionaceae. Given the increasing prevalence of multi-drug resistance in pathogenic vibrios, there is a need for new targets and drugs to combat these pathogens. Here, we carried out a high throughput cell-based screen to find small molecule inhibitors of RctB. We identified a compound that blocked growth of an E. coli strain bearing an rctB-dependent plasmid but did not influence growth of E. coli lacking this plasmid. This compound, designated vibrepin, had potent cidal activity against V. cholerae and inhibited the growth of all vibrio species tested. Vibrepin blocked RctB oriCII unwinding, apparently by promoting formation of large non-functional RctB complexes. Although vibrepin also appears to have targets other than RctB, our findings suggest that RctB is an attractive target for generation of novel antibiotics that only block growth of vibrios. Vibrio-specific agents, unlike antibiotics currently used in clinical practice, will not engender resistance in the normal human flora or in non-vibrio environmental microorganisms.


Assuntos
Anti-Infecciosos/farmacologia , Proteínas de Bactérias/efeitos dos fármacos , Cromossomos Bacterianos/genética , DNA Helicases/efeitos dos fármacos , Transativadores/efeitos dos fármacos , Vibrionaceae/genética , Farmacorresistência Bacteriana/efeitos dos fármacos
8.
Proc Natl Acad Sci U S A ; 105(30): 10577-82, 2008 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-18647828

RESUMO

Vibrio cholerae, the agent of cholera, has two circular chromosomes. In bacteria that contain a single chromosome, initiation of chromosome DNA replication is mediated by DnaA, a AAA+ ATPase that unwinds the origin of replication. There is little knowledge regarding initiation of chromosome replication in bacteria with more than one chromosome. Here, we purified V. cholerae DnaA and RctB, which have been implicated in the replication of V. cholerae chromosome II, and characterized their activities in vitro. We found that RctB has origin-specific unwinding activity and can melt the origin of chromosome II (oriCIIvc) but not the origin of chromosome I (oriCIvc); conversely, DnaA promoted the unwinding of oriCIvc and not oriCIIvc. The activity of DnaA and several plasmid initiator proteins is stimulated by ATP binding. We found that RctB bound and hydrolyzed ATP even though RctB lacks any apparent ATP-binding motifs. However, we unexpectedly found that ATP inhibited the oriCIIvc binding activity of RctB, suggesting that the ATP-bound form of RctB cannot initiate replication of chromosome II. Supporting this idea, we identified an RctB mutant that does not bind ATP and found that expression of this ATP-blind RctB mutant in V. cholerae leads to significant overinitiation of chromosome II and marked inhibition of V. cholerae growth. These observations suggest that the rules that license the replication of the two V. cholerae chromosomes differ.


Assuntos
Trifosfato de Adenosina/química , Cromossomos Bacterianos/ultraestrutura , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Vibrio cholerae/genética , Difosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Hidrólise , Modelos Genéticos , Mutação , Origem de Replicação
9.
J Bacteriol ; 188(17): 6419-24, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16923911

RESUMO

Although the two Vibrio cholerae chromosomes initiate replication in a coordinated fashion, we show here that each chromosome appears to have a specific replication initiator. DnaA overproduction promoted overinitiation of chromosome I and not chromosome II. In contrast, overproduction of RctB, a protein that binds to the origin of replication of chromosome II, promoted overinitiation of chromosome II and not chromosome I.


Assuntos
Proteínas de Bactérias/fisiologia , Cromossomos Bacterianos/genética , Replicação do DNA , Vibrio cholerae/genética , Cromossomos Bacterianos/metabolismo , Proteínas de Ligação a DNA/fisiologia
10.
J Bacteriol ; 188(2): 789-93, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16385068

RESUMO

The RctB protein binds to the origin of replication of Vibrio cholerae chromosome II (chrII) and is required for oriCIIVc-based replication. Here, we found that RctB acts as an autorepressor, inhibiting rctB transcription. Integration host factor promotes rctB transcription, while Dam and DnaA, factors required for replication of both V. cholerae chromosomes, influence RctB autorepression. Thus, RctB appears to regulate chrII replication as both an initiator and a transcription repressor, and its synthesis is modulated by factors that govern replication of both chromosomes.


Assuntos
Cromossomos Bacterianos/metabolismo , Proteínas Repressoras/genética , Vibrio cholerae/genética , Cromossomos Bacterianos/genética , Replicação do DNA , Origem de Replicação , Fatores de Transcrição , Sítio de Iniciação de Transcrição
11.
Mol Microbiol ; 57(3): 678-90, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16045613

RESUMO

Translesion synthesis (TLS) across damaged DNA bases is most often carried out by the ubiquitous error-prone DNA polymerases of the Y-family. Bacillus subtilis encodes two Y-polymerases, Pol Y1 and Pol Y2, that mediate TLS resulting in spontaneous and ultraviolet light (UV)-induced mutagenesis respectively. Here we show that TLS is a bipartite dual polymerase process in B. subtilis, involving not only the Y-polymerases but also the A-family polymerase, DNA polymerase I (Pol I). Both the spontaneous and the UV-induced mutagenesis are abolished in Pol I mutants affected solely in the polymerase catalytic site. Physical interactions between Pol I and either of the Pol Y polymerases, as well as formation of a ternary complex between Pol Y1, Pol I and the beta-clamp, were detected by yeast two- and three-hybrid assays, supporting the model of a functional coupling between the A- and Y-family polymerases in TLS. We suggest that the Pol Y carries the synthesis across the lesion, and Pol I takes over to extend the synthesis until the functional replisome resumes replication. This key role of Pol I in TLS uncovers a new function of the A-family DNA polymerases.


Assuntos
Bacillus subtilis/enzimologia , Dano ao DNA , DNA Polimerase I/metabolismo , Reparo do DNA , DNA Bacteriano/biossíntese , Regulação Bacteriana da Expressão Gênica , Bacillus subtilis/genética , Bacillus subtilis/efeitos da radiação , Replicação do DNA , DNA Bacteriano/genética , DNA Polimerase Dirigida por DNA/classificação , DNA Polimerase Dirigida por DNA/metabolismo , Mutagênese , Técnicas do Sistema de Duplo-Híbrido , Raios Ultravioleta
12.
Mol Microbiol ; 54(2): 439-51, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15469515

RESUMO

Translesional DNA polymerases form a large family of structurally related proteins, known as the Y-polymerases. Bacillus subtilis encodes two Y-polymerases, referred herewith as Pol Y1 and Pol Y2. Pol Y1 was expressed constitutively and did not mediate UV mutagenesis. Pol Y1 overexpression increased spontaneous mutagenesis. This effect depended on Pol Y1 polymerase activity, Pol Y1 interaction with the beta-clamp, and did not require the presence of the RecA protein. In addition, Pol Y1 overexpression delayed cell growth at low temperature. The growth delay was mediated by Pol Y1 interaction with the beta-clamp but not by its polymerase activity, suggesting that an excess of Pol Y1 in the cell could sequester the beta-clamp. In contrast, Pol Y2 was expressed during the SOS response, and, in its absence, UV-induced mutagenesis was abolished. Upon Pol Y2 overproduction, both UV-induced and spontaneous mutagenesis were stimulated, and both depended on the Pol Y2 polymerase activity. However, UV mutagenesis did not appear to require the interaction of Pol Y2 with the beta-clamp whereas spontaneous mutagenesis did. In addition, Pol Y2-mediated spontaneous mutagenesis required the presence of RecA. Together, these results show that the regulation and the genetic requirements of the two B. subtilis Y-polymerases are different, indicating that they fulfil distinct biological roles. Remarkably, Pol Y1 appears to exhibit a mutator activity similar to that of Escherichia coli Pol IV, as well as an E. coli UmuD-related function in growth delay. Pol Y2 exhibits an E. coli Pol V-like mutator activity, but probably acts as a single polypeptide to bypass UV lesions. Thus, B. subtilis Pol Y1 and Pol Y2 exhibit distinctive features from the E. coli Y-polymerases, indicating that different bacteria have adapted different solutions to deal with the lesions in their genetic material.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/genética , DNA Polimerase Dirigida por DNA/genética , Motivos de Aminoácidos , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Dano ao DNA , DNA Bacteriano/efeitos da radiação , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Genótipo , Mutação , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Temperatura , Raios Ultravioleta
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