RESUMEN

The proper maintenance of genetic material is essential for the survival of living organisms. One of the main safeguards of genome stability is homologous recombination involved in the faithful repair of DNA double-strand breaks, the restoration of collapsed replication forks, and the bypass of replication barriers. Homologous recombination relies on the formation of Rad51 nucleoprotein filaments which are responsible for the homology-based interactions between DNA strands. Here, we demonstrate that without the regulation of these filaments by Srs2 and Rad54, which are known to remove Rad51 from single-stranded and double-stranded DNA, respectively, the filaments strongly inhibit damage-associated DNA synthesis during DNA repair. Furthermore, this regulation is essential for cell survival under normal growth conditions, as in the srs2Δ rad54Δ mutants, unregulated Rad51 nucleoprotein filaments cause activation of the DNA damage checkpoint, formation of mitotic bridges, and loss of genetic material. These genome instability features may stem from the problems at stalled replication forks as the lack of Srs2 and Rad54 in the presence of Rad51 nucleoprotein filaments impedes cell recovery from replication stress. This study demonstrates that the timely and efficient disassembly of recombination machinery is essential for genome maintenance and cell survival.

RESUMEN

From their synthesis in the nucleus to their degradation in the cytoplasm, all mRNAs have the same objective, which is to translate the DNA-stored genetic information into functional proteins at the proper time and location. To this end, many proteins are generally associated with mRNAs as soon as transcription takes place in the nucleus to organize spatiotemporal regulation of the gene expression in cells. Here we reviewed how YB-1 (YBX1 gene), one of the major mRNA-binding proteins in the cytoplasm, packaged mRNAs into either compact or extended linear nucleoprotein mRNPs. Interestingly the structural plasticity of mRNPs coordinated by YB-1 could provide means for the contextual regulation of mRNA translation. Posttranslational modification of YB-1, notably in the long unstructured YB-1 C-terminal domain (CTD), and/or the protein partners of YB-1 may play a key role in activation/inactivation of mRNPs in the cells notably in response to cellular stress.

Asunto(s)

Biosíntesis de Proteínas , Gránulos de Estrés , Citoplasma/metabolismo , Procesamiento Proteico-Postraduccional , ARN Mensajero/metabolismo

RESUMEN

The recombinases present in the all kingdoms in nature play a crucial role in DNA metabolism processes such as replication, repair, recombination and transcription. However, till date, the role of RecA in the deadly foodborne pathogen Listeria monocytogenes remains unknown. In this study, the authors show that L. monocytogenes expresses recA more than two-fold in vivo upon exposure to the DNA damaging agents, methyl methanesulfonate and ultraviolet radiation. The purified L. monocytogenes RecA protein show robust binding to single stranded DNA. The RecA is capable of forming displacement loop and hydrolyzes ATP, whereas the mutant LmRecAK70A fails to hydrolyze ATP, showing conserved walker A and B motifs. Interestingly, L. monocytogenes RecA and LmRecAK70A perform the DNA strand transfer activity, which is the hallmark feature of RecA protein with an oligonucleotide-based substrate. Notably, L. monocytogenes RecA readily cleaves L. monocytogenes LexA, the SOS regulon and protects the presynaptic filament from the exonuclease I activity. Altogether, this study provides the first detailed characterization of L. monocytogenes RecA and presents important insights into the process of homologous recombination in the gram-positive foodborne bacteria L. monocytogenes.

Asunto(s)

Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , Recombinación Homóloga , Listeria monocytogenes/enzimología , Respuesta SOS en Genética , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/metabolismo

RESUMEN

Bacterial RecA plays an important role in the evaluation of antibiotic resistance via stress-induced DNA repair mechanism; SOS response. Accordingly, RecA became an important therapeutic target against antimicrobial resistance. Small molecule inhibitors of RecA may prevent adaptation of antibiotic resistance mutations and the emergence of antimicrobial resistance. In our study, we observed that phenolic compound p-Coumaric acid as potent RecA inhibitor. It inhibited RecA driven biochemical activities in vitro such as ssDNA binding, strand exchange, ATP hydrolysis and RecA coprotease activity of E. coli and L. monocytogenes RecA proteins. The mechanism underlying such inhibitory action of p-Coumaric acid involves its ability to interfere with the DNA binding domain of RecA protein. p-Coumaric acid also potentiates the activity of ciprofloxacin by inhibiting drastic cell survival of L. monocytogenes as well as filamentation process; the bacteria defensive mechanism in response to DNA damage. Additionally, it also blocked the ciprofloxacin induced RecA expression leading to suppression of SOS response in L. monocytogenes. These findings revealed that p-Coumaric acid is a potent RecA inhibitor, and can be used as an adjuvant to the existing antibiotics which not only enhance the shelf-life but also slow down the emergence of antibiotic resistance in bacteria.

Asunto(s)

Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Listeria monocytogenes/efectos de los fármacos , Propionatos/farmacología , Rec A Recombinasas/antagonistas & inhibidores , Respuesta SOS en Genética/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Ciprofloxacina/farmacología , Ácidos Cumáricos , Reparación del ADN/efectos de los fármacos , ADN Bacteriano/antagonistas & inhibidores , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Farmacorresistencia Bacteriana Múltiple/genética , Sinergismo Farmacológico , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Expresión Génica , Hidrólisis/efectos de los fármacos , Listeria monocytogenes/genética , Listeria monocytogenes/crecimiento & desarrollo , Listeria monocytogenes/metabolismo , Pruebas de Sensibilidad Microbiana , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismo , Recombinación Genética/efectos de los fármacos

RESUMEN

The meiosis-specific recombinase, DMC1, is important for the generation of haploids during meiosis. DMC1 forms a helical nucleoprotein filament on ssDNA overhangs located at the processed double-stranded DNA break. The DMC1 filament performs a search for homology in homologous chromosome. Once homology is located, the DMC1 filament strand invades the homologous chromosome forming a displacement loop (D-loop). These connections are needed for accurate segregation to occur later in meiosis. Because DMC1 requires numerous accessory factors and specific ionic conditions to participate in this DNA repair process, in vitro assays were developed to understand how these accessory factors influence the biochemical properties of hDMC1. This chapter describes a method that can be used to investigate the stability of the human DMC1 nucleoprotein filament under various conditions and provides insight into an important early stage in DNA double-strand break repair by homologous recombination during meiosis.

Asunto(s)

Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Nucleoproteínas/metabolismo , Recombinasas/metabolismo , Reparación del ADN por Recombinación , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/aislamiento & purificación , Roturas del ADN de Doble Cadena , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Meiosis/genética , Nucleoproteínas/genética , Nucleoproteínas/aislamiento & purificación , Estabilidad Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Recombinasas/genética , Recombinasas/aislamiento & purificación

RESUMEN

The simplest forms of mutations, base substitutions, typically have negative consequences, aside from their existential role in evolution and fitness. Hypermutations, mutations on steroids, occurring at frequencies of 10(-2) -10(-4) per base pair, straddle a domain between fitness and death, depending on the presence or absence of regulatory constraints. Two facets of hypermutation, one in Escherichia coli involving DNA polymerase V (pol V), the other in humans, involving activation-induced deoxycytidine deaminase (AID) are portrayed. Pol V is induced as part of the DNA-damage-induced SOS regulon, and is responsible for generating the lion's share of mutations when catalyzing translesion DNA synthesis (TLS). Four regulatory mechanisms, temporal, internal, conformational, and spatial, activate pol V to copy damaged DNA and then deactivate it. On the flip side of the coin, SOS-induced pols V, IV, and II mutate undamaged DNA, thus providing genetic diversity heightening long-term survival and evolutionary fitness. Fitness in humans is principally the domain of a remarkably versatile immune system marked by somatic hypermutations (SHM) in immunoglobulin variable (IgV) regions that ensure antibody (Ab) diversity. AID initiates SHM by deaminating C → U, favoring hot WRC (W = A/T, R = A/G) motifs. Since there are large numbers of trinucleotide motif targets throughout IgV, AID must exercise considerable catalytic restraint to avoid attacking such sites repeatedly, which would otherwise compromise diversity. Processive, random, and inefficient AID-catalyzed dC deamination simulates salient features of SHM, yet generates B-cell lymphomas when working at the wrong time in the wrong place. Environ. Mol. Mutagen. 57:421-434, 2016. © 2016 Wiley Periodicals, Inc.

Asunto(s)

Citidina Desaminasa/genética , ADN Polimerasa Dirigida por ADN/genética , Proteínas de Escherichia coli/genética , Región Variable de Inmunoglobulina/genética , Mutación , Hipermutación Somática de Inmunoglobulina , Escherichia coli/genética , Aptitud Genética/inmunología , Humanos , Modelos Genéticos , Tasa de Mutación , Respuesta SOS en Genética , Moldes Genéticos

RESUMEN

My career pathway has taken a circuitous route, beginning with a Ph.D. degree in electrical engineering from The Johns Hopkins University, followed by five postdoctoral years in biology at Hopkins and culminating in a faculty position in biological sciences at the University of Southern California. My startup package in 1973 consisted of $2,500, not to be spent all at once, plus an ancient Packard scintillation counter that had a series of rapidly flashing light bulbs to indicate a radioactive readout in counts/minute. My research pathway has been similarly circuitous. The discovery of Escherichia coli DNA polymerase V (pol V) began with an attempt to identify the mutagenic DNA polymerase responsible for copying damaged DNA as part of the well known SOS regulon. Although we succeeded in identifying a DNA polymerase, one that was induced as part of the SOS response, we actually rediscovered DNA polymerase II, albeit in a new role. A decade later, we discovered a new polymerase, pol V, whose activity turned out to be regulated by bound molecules of RecA protein and ATP. This Reflections article describes our research trajectory, includes a review of key features of DNA damage-induced SOS mutagenesis leading us to pol V, and reflects on some of the principal researchers who have made indispensable contributions to our efforts.

Asunto(s)

ADN Polimerasa Dirigida por ADN/historia , Proteínas de Escherichia coli/historia , Escherichia coli/enzimología , Biología Molecular/historia , Rec A Recombinasas/historia , ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Rec A Recombinasas/metabolismo

RESUMEN

MuB is an ATP-dependent nonspecific DNA-binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. Mechanistic understanding of MuB function has previously been hindered by MuB's poor solubility. Here we combine bioinformatic, mutagenic, biochemical, and electron microscopic analyses to unmask the structure and function of MuB. We demonstrate that MuB is an ATPase associated with diverse cellular activities (AAA+ ATPase) and forms ATP-dependent filaments with or without DNA. We also identify critical residues for MuB's ATPase, DNA binding, protein polymerization, and MuA interaction activities. Using single-particle electron microscopy, we show that MuB assembles into a helical filament, which binds the DNA in the axial channel. The helical parameters of the MuB filament do not match those of the coated DNA. Despite this protein-DNA symmetry mismatch, MuB does not deform the DNA duplex. These findings, together with the influence of MuB filament size on strand-transfer efficiency, lead to a model in which MuB-imposed symmetry transiently deforms the DNA at the boundary of the MuB filament and results in a bent DNA favored by MuA for transposition.

Asunto(s)

Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Bacteriófago mu/enzimología , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Bacteriófago mu/genética , Sitios de Unión/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Imagenología Tridimensional , Microscopía Electrónica de Transmisión , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Multimerización de Proteína/genética , Homología de Secuencia de Aminoácido , Transposasas/genética , Transposasas/metabolismo , Proteínas Virales/genética

RESUMEN

Transposition target immunity is a phenomenon observed in some DNA transposons that are able to distinguish the host chromosome from their own DNA sequence, thus avoiding self-destructive insertions. The first molecular insight into target selection and immunity mechanisms came from the study of phage Mu transposition, which uses the protein MuB as a barrier to self-insertion. MuB is an ATP-dependent non-specific DNA binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. However, a detailed mechanistic understanding of MuB functioning was hindered by the poor solubility of the MuB-ATP complexes. Here we comment on the recent discovery that MuB is an AAA+ ATPase that upon ATP binding assembles into helical filaments that coat the DNA. Remarkably, the helical parameters of the MuB filament do not match those of the bound DNA. This intriguing mismatch symmetry led us to propose a model on how MuB targets DNA for transposition, favoring DNA bending and recognition by the transposase at the filament edge. We also speculate on a different protective role of MuB during immunity, where filament stickiness could favor the condensation of the DNA into a compact state that occludes it from the transposase.