Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 12 de 12
Filtrar
Más filtros











Base de datos
Intervalo de año de publicación
1.
Biochem Biophys Res Commun ; 716: 150009, 2024 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-38697010

RESUMEN

The SOS response is a condition that occurs in bacterial cells after DNA damage. In this state, the bacterium is able to reсover the integrity of its genome. Due to the increased level of mutagenesis in cells during the repair of DNA double-strand breaks, the SOS response is also an important mechanism for bacterial adaptation to the antibiotics. One of the key proteins of the SOS response is the SMC-like protein RecN, which helps the RecA recombinase to find a homologous DNA template for repair. In this work, the localization of the recombinant RecN protein in living Escherichia coli cells was revealed using fluorescence microscopy. It has been shown that the RecN, outside the SOS response, is predominantly localized at the poles of the cell, and in dividing cells, also localized at the center. Using in vitro methods including fluorescence microscopy and optical tweezers, we show that RecN predominantly binds single-stranded DNA in an ATP-dependent manner. RecN has both intrinsic and single-stranded DNA-stimulated ATPase activity. The results of this work may be useful for better understanding of the SOS response mechanism and homologous recombination process.


Asunto(s)
ADN Bacteriano , Escherichia coli , Microscopía Fluorescente , Imagen Individual de Molécula , Microscopía Fluorescente/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Imagen Individual de Molécula/métodos , ADN Bacteriano/metabolismo , ADN Bacteriano/genética , Respuesta SOS en Genética , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Unión Proteica , Rec A Recombinasas/metabolismo , Rec A Recombinasas/genética , Pinzas Ópticas
2.
Int J Mol Sci ; 23(18)2022 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-36142700

RESUMEN

Serratia proteamaculans synthesizes the intracellular metalloprotease protealysin. This work was aimed at searching for bacterial substrates of protealysin among the proteins responsible for replication and cell division. We have shown that protealysin unlimitedly cleaves the SOS response protein RecA. Even 20% of the cleaved RecA in solution appears to be incorporated into the polymer of uncleaved monomers, preventing further polymerization and inhibiting RecA ATPase activity. Transformation of Escherichia coli with a plasmid carrying the protealysin gene reduces the bacterial UV survival up to 10 times. In addition, the protealysin substrate is the FtsZ division protein, found in both E. coli and Acholeplasma laidlawii, which is only 51% identical to E. coli FtsZ. Protealysin cleaves FtsZ at the linker between the globular filament-forming domain and the C-terminal peptide that binds proteins on the bacterial membrane. Thus, cleavage of the C-terminal segment by protealysin can lead to the disruption of FtsZ's attachment to the membrane, and thereby inhibit bacterial division. Since the protealysin operon encodes not only the protease, but also its inhibitor, which is typical for the system of interbacterial competition, we assume that in the case of penetration of protealysin into neighboring bacteria that do not synthesize a protealysin inhibitor, cleavage of FtsZ and RecA by protealysin may give S. proteamaculans an advantage in interbacterial competition.


Asunto(s)
Proteínas Bacterianas , Proteínas de Escherichia coli , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Tareas del Hogar , Metaloproteasas/metabolismo , Péptido Hidrolasas/metabolismo , Péptidos/química , Polímeros/metabolismo
3.
Comput Struct Biotechnol J ; 19: 777-783, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33552448

RESUMEN

Antibiotic resistance is acquired in response to antibiotic therapy by activating SOS-depended mutagenesis and horizontal gene transfer pathways. Compounds able to inhibit SOS response are extremely important to develop new combinatorial strategies aimed to block mutagenesis. The regulators of homologous recombination involved in the processes of DNA repair should be considered as potential targets for blocking. This review highlights the current knowledge of the protein targets for the evolution of antibiotic resistance and the inhibitory effects of some new compounds on this pathway.

4.
Int J Mol Sci ; 21(19)2020 Oct 07.
Artículo en Inglés | MEDLINE | ID: mdl-33036395

RESUMEN

Deinococcus radiodurans (Dr) has one of the most robust DNA repair systems, which is capable of withstanding extreme doses of ionizing radiation and other sources of DNA damage. DrRecA, a central enzyme of recombinational DNA repair, is essential for extreme radioresistance. In the presence of ATP, DrRecA forms nucleoprotein filaments on DNA, similar to other bacterial RecA and eukaryotic DNA strand exchange proteins. However, DrRecA catalyzes DNA strand exchange in a unique reverse pathway. Here, we study the dynamics of DrRecA filaments formed on individual molecules of duplex and single-stranded DNA, and we follow conformational transitions triggered by ATP hydrolysis. Our results reveal that ATP hydrolysis promotes rapid DrRecA dissociation from duplex DNA, whereas on single-stranded DNA, DrRecA filaments interconvert between stretched and compressed conformations, which is a behavior shared by E. coli RecA and human Rad51. This indicates a high conservation of conformational switching in nucleoprotein filaments and suggests that additional factors might contribute to an inverse pathway of DrRecA strand exchange.


Asunto(s)
Adenosina Trifosfato/química , Proteínas Bacterianas/química , Deinococcus/enzimología , Modelos Moleculares , Conformación Molecular , Rec A Recombinasas/química , Imagen Individual de Molécula , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Hidrólisis , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Rec A Recombinasas/metabolismo , Imagen Individual de Molécula/métodos
5.
FEBS Lett ; 594(21): 3464-3476, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32880917

RESUMEN

The RecA protein plays a key role in bacterial homologous recombination (HR) and acts through assembly of long helical filaments around single-stranded DNA in the presence of ATP. Large-scale conformational changes induced by ATP hydrolysis result in transitions between stretched and compressed forms of the filament. Here, using a single-molecule approach, we show that compressed RecA nucleoprotein filaments can exist in two distinct interconvertible states depending on the presence of ADP in the monomer-monomer interface. Binding of ADP promotes cooperative conformational transitions and directly affects mechanical properties of the filament. Our findings reveal that RecA nucleoprotein filaments are able to continuously cycle between three mechanically distinct states that might have important implications for RecA-mediated processes of HR.


Asunto(s)
ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Rec A Recombinasas/química , Rec A Recombinasas/metabolismo , Imagen Individual de Molécula , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Escherichia coli
6.
Nucleic Acids Res ; 45(16): 9788-9796, 2017 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-28934502

RESUMEN

The RecX protein, a very active natural RecA protein inhibitor, can completely disassemble RecA filaments at nanomolar concentrations that are two to three orders of magnitude lower than that of RecA protein. Based on the structure of RecX protein complex with the presynaptic RecA filament, we designed a short first in class α-helical peptide that both inhibits RecA protein activities in vitro and blocks the bacterial SOS-response in vivo. The peptide was designed using SEQOPT, a novel method for global sequence optimization of protein α-helices. SEQOPT produces artificial peptide sequences containing only 20 natural amino acids with the maximum possible conformational stability at a given pH, ionic strength, temperature, peptide solubility. It also accounts for restrictions due to known amino acid residues involved in stabilization of protein complexes under consideration. The results indicate that a few key intermolecular interactions inside the RecA protein presynaptic complex are enough to reproduce the main features of the RecX protein mechanism of action. Since the SOS-response provides a major mechanism of bacterial adaptation to antibiotics, these results open new ways for the development of antibiotic co-therapy that would not cause bacterial resistance.


Asunto(s)
Péptidos/química , Péptidos/farmacología , Rec A Recombinasas/antagonistas & inhibidores , Respuesta SOS en Genética/efectos de los fármacos , Dicroismo Circular , ADN/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Escherichia coli/efectos de la radiación , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformación Proteica , Estabilidad Proteica , Rec A Recombinasas/química , Rec A Recombinasas/metabolismo , Respuesta SOS en Genética/efectos de la radiación , Rayos Ultravioleta
7.
PLoS One ; 11(4): e0154137, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27124470

RESUMEN

The RecA recombinase of Escherichia coli has not evolved to optimally promote DNA pairing and strand exchange, the key processes of recombinational DNA repair. Instead, the recombinase function of RecA protein represents an evolutionary compromise between necessary levels of recombinational DNA repair and the potentially deleterious consequences of RecA functionality. A RecA variant, RecA D112R, promotes conjugational recombination at substantially enhanced levels. However, expression of the D112R RecA protein in E. coli results in a reduction in cell growth rates. This report documents the consequences of the substantial selective pressure associated with the RecA-mediated hyperrec phenotype. With continuous growth, the deleterious effects of RecA D112R, along with the observed enhancements in conjugational recombination, are lost over the course of 70 cell generations. The suppression reflects a decline in RecA D112R expression, associated primarily with a deletion in the gene promoter or chromosomal mutations that decrease plasmid copy number. The deleterious effects of RecA D112R on cell growth can also be negated by over-expression of the RecX protein from Neisseria gonorrhoeae. The effects of the RecX proteins in vivo parallel the effects of the same proteins on RecA D112R filaments in vitro. The results indicate that the toxicity of RecA D112R is due to its persistent binding to duplex genomic DNA, creating barriers for other processes in DNA metabolism. A substantial selective pressure is generated to suppress the resulting barrier to growth.


Asunto(s)
Proteínas Bacterianas/genética , ADN Bacteriano/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Rec A Recombinasas/genética , Sustitución de Aminoácidos , Arginina/metabolismo , Ácido Aspártico/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Conjugación Genética , ADN Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Genotipo , Mutación , Neisseria gonorrhoeae/química , Fenotipo , Plásmidos/química , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Rec A Recombinasas/metabolismo , Reparación del ADN por Recombinación
8.
FEBS Lett ; 588(6): 948-55, 2014 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-24530684

RESUMEN

Using molecular modeling techniques we have built the full atomic structure and performed molecular dynamics simulations for the complexes formed by Escherichia coli RecX protein with a single-stranded oligonucleotide and with RecA presynaptic filament. Based on the modeling and SANS experimental data a sandwich-like filament structure formed two chains of RecX monomers bound to the opposite sides of the single stranded DNA is proposed for RecX::ssDNA complex. The model for RecX::RecA::ssDNA include RecX binding into the grove of RecA::ssDNA filament that occurs mainly via Coulomb interactions between RecX and ssDNA. Formation of RecX::RecA::ssDNA filaments in solution was confirmed by SANS measurements which were in agreement with the spectra computed from the molecular dynamics simulations.


Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli , Simulación de Dinámica Molecular , Rec A Recombinasas/química , ADN de Cadena Simple/química , Difracción de Neutrones , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Dispersión del Ángulo Pequeño , Soluciones
9.
Mol Microbiol ; 78(6): 1523-38, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21143322

RESUMEN

The wild-type Escherichia coli RecA protein is a recombinase platform with unrealized recombination potential. We have explored the factors affecting recombination during conjugation with a quantitative assay. Regulatory proteins that affect RecA function have the capacity to increase or decrease recombination frequencies by factors up to sixfold. Autoinhibition by the RecA C-terminus can affect recombination frequency by factors up to fourfold. The greatest changes in recombination frequency measured here are brought about by point mutations in the recA gene. RecA variants can increase recombination frequencies by more than 50-fold. The RecA protein thus possesses an inherently broad functional range. The RecA protein of E. coli (EcRecA) is not optimized for recombination function. Instead, much of the recombination potential of EcRecA is structurally suppressed, probably reflecting cellular requirements. One point mutation in EcRecA with a particularly dramatic effect on recombination frequency, D112R, exhibits an enhanced capacity to load onto SSB-coated ssDNA, overcome the effects of regulatory proteins such as PsiB and RecX, and to pair homologous DNAs. Comparisons of key RecA protein mutants reveal two components to RecA recombination function - filament formation and the inherent DNA pairing activity of the formed filaments.


Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Rec A Recombinasas/química , Rec A Recombinasas/metabolismo , Recombinación Genética , Secuencias de Aminoácidos , Conjugación Genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Evolución Molecular , Regulación Bacteriana de la Expresión Génica , Mutación , Rec A Recombinasas/genética
10.
J Bacteriol ; 190(8): 3036-45, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18296520

RESUMEN

RecAX53 is a chimeric variant of the Escherichia coli RecA protein (RecAEc) that contains a part of the central domain of Pseudomonas aeruginosa RecA (RecAPa), encompassing a region that differs from RecAEc at 12 amino acid positions. Like RecAPa, this chimera exhibits hyperrecombination activity in E. coli cells, increasing the frequency of recombination exchanges per DNA unit length (FRE). RecAX53 confers the largest increase in FRE observed to date. The contrasting properties of RecAX53 and RecAPa are manifested by in vivo differences in the dependence of the FRE value on the integrity of the mutS gene and thus in the ratio of conversion and crossover events observed among their hyperrecombination products. In strains expressing the RecAPa or RecAEc protein, crossovers are the main mode of hyperrecombination. In contrast, conversions are the primary result of reactions promoted by RecAX53. The biochemical activities of RecAX53 and its ancestors, RecAEc and RecAPa, have been compared. Whereas RecAPa generates a RecA presynaptic complex (PC) that is more stable than that of RecAEc, RecAX53 produces a more dynamic PC (relative to both RecAEc and RecAPa). The properties of RecAX53 result in a more rapid initiation of the three-strand exchange reaction but an inability to complete the four-strand transfer. This indicates that RecAX53 can form heteroduplexes rapidly but is unable to convert them into crossover configurations. A more dynamic RecA activity thus translates into an increase in conversion events relative to crossovers.


Asunto(s)
Escherichia coli/enzimología , Pseudomonas aeruginosa/enzimología , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Recombinación Genética , Intercambio Genético , ADN Bacteriano/metabolismo , Reordenamiento Génico , Cinética
11.
J Bacteriol ; 188(16): 5812-20, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16885449

RESUMEN

In Escherichia coli, a relatively low frequency of recombination exchanges (FRE) is predetermined by the activity of RecA protein, as modulated by a complex regulatory program involving both autoregulation and other factors. The RecA protein of Pseudomonas aeruginosa (RecA(Pa)) exhibits a more robust recombinase activity than its E. coli counterpart (RecA(Ec)). Low-level expression of RecA(Pa) in E. coli cells results in hyperrecombination (an increase of FRE) even in the presence of RecA(Ec). This genetic effect is supported by the biochemical finding that the RecA(Pa) protein is more efficient in filament formation than RecA K72R, a mutant protein with RecA(Ec)-like DNA-binding ability. Expression of RecA(Pa) also partially suppresses the effects of recF, recO, and recR mutations. In concordance with the latter, RecA(Pa) filaments initiate recombination equally from both the 5' and 3' ends. Besides, these filaments exhibit more resistance to disassembly from the 5' ends that makes the ends potentially appropriate for initiation of strand exchange. These comparative genetic and biochemical characteristics reveal that multiple levels are used by bacteria for a programmed regulation of their recombination activities.


Asunto(s)
Escherichia coli/genética , Escherichia coli/metabolismo , Pseudomonas aeruginosa/metabolismo , Rec A Recombinasas/metabolismo , ADN Bacteriano/genética , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Mutación , Pseudomonas aeruginosa/genética , Rec A Recombinasas/genética , Recombinación Genética/genética , Recombinación Genética/fisiología
12.
Genetics ; 163(4): 1243-54, 2003 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-12702672

RESUMEN

The frequency of recombinational exchanges (FRE) that disrupt co-inheritance of transferred donor markers in Escherichia coli Hfr by F(-) crosses differs by up to a factor of two depending on physiological factors and culture conditions. Under standard conditions we found FRE to be 5.01 +/- 0.43 exchanges per 100-min units of DNA length for wild-type strains of the AB1157 line. Using these conditions we showed a cumulative effect of various mutations on FRE. Constitutive SOS expression by lexA gene inactivation (lexA71::Tn5) and recA gene mutation (recA730) showed, respectively, approximately 4- and 7-fold increases of FRE. The double lexA71 recA730 combination gave an approximately 17-fold increase in FRE. Addition of mutS215::Tn10, inactivating the mismatch repair system, to the double lexA recA mutant increased FRE to approximately 26-fold above wild-type FRE. Finally, we showed that another recA mutation produced as much SOS expression as recA730 but increased FRE only 3-fold. We conclude that three factors contribute to normally low FRE under standard conditions: repression of the LexA regulon, the properties of wild-type RecA protein, and a functioning MutSHL mismatch repair system. We discuss mechanisms by which the lexA, recA, and mutS mutations may elevate FRE cumulatively to obtain hyperrecombination.


Asunto(s)
Proteínas Bacterianas/genética , Conjugación Genética , Reparación del ADN , Proteínas de Unión al ADN , Escherichia coli/genética , Rec A Recombinasas/metabolismo , Serina Endopeptidasas/genética , Adenosina Trifosfatasas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN , Rec A Recombinasas/genética , Serina Endopeptidasas/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA