RESUMO
Plasmids play a major role in rapid adaptation of bacteria by facilitating horizontal transfer of diverse genes, most notably those conferring antibiotic resistance. While most plasmids that replicate in a broad range of bacteria also persist well in diverse hosts, there are exceptions that are poorly understood. We investigated why a broad-host range plasmid, pBP136, originally found in clinical Bordetella pertussis isolates, quickly became extinct in laboratory Escherichia coli populations. Through experimental evolution we found that inactivation of a previously uncharacterized plasmid gene, upf31, drastically improved plasmid maintenance in E. coli. This gene inactivation resulted in decreased transcription of the global plasmid regulators (korA, korB, and korC) and numerous genes in their regulons. It also caused transcriptional changes in many chromosomal genes primarily related to metabolism. In silico analyses suggested that the change in plasmid transcriptome may be initiated by Upf31 interacting with the plasmid regulator KorB. Expression of upf31 in trans negatively affected persistence of pBP136Δupf31 as well as the closely related archetypal IncP-1ß plasmid R751, which is stable in E. coli and natively encodes a truncated upf31 allele. Our results demonstrate that while the upf31 allele in pBP136 might advantageously modulate gene expression in its original host, B. pertussis, it has harmful effects in E. coli. Thus, evolution of a single plasmid gene can change the range of hosts in which that plasmid persists, due to effects on the regulation of plasmid gene transcription.
RESUMO
Central to the universal process of recombination, RecA family proteins form nucleoprotein filaments to catalyze production of heteroduplex DNA between substrate ssDNAs and template dsDNAs. ATP binding assists the filament in assuming the necessary conformation for forming heteroduplex DNA, but hydrolysis is not required. ATP hydrolysis has two identified roles which are not universally conserved: promotion of filament dissociation and enhancing flexibility of the filament. In this work, we examine ATP utilization of the RecA family recombinase SsoRadA from Saccharolobus solfataricus to determine its function in recombinase-mediated heteroduplex DNA formation. Wild-type SsoRadA protein and two ATPase mutant proteins were evaluated for the effects of three divalent metal cofactors. We found that unlike other archaeal RadA proteins, SsoRadA-mediated strand exchange is not enhanced by Ca2+. Instead, the S. solfataricus recombinase can utilize Mn2+ to stimulate strand invasion and reduce ADP-binding stability. Additionally, reduction of SsoRadA ATPase activity by Walker Box mutation or cofactor alteration resulted in a loss of large, complete strand exchange products. Depletion of ADP was found to improve initial strand invasion but also led to a similar loss of large strand exchange events. Our results indicate that overall, SsoRadA is distinct in its use of divalent cofactors but its activity with Mn2+ shows similarity to human RAD51 protein with Ca2+.
Assuntos
Cálcio , Sulfolobus solfataricus , Humanos , Cálcio/metabolismo , Ácidos Nucleicos Heteroduplexes/metabolismo , Recombinases Rec A/metabolismo , Sulfolobus solfataricus/genética , Sulfolobus solfataricus/metabolismo , Recombinases/metabolismo , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismoRESUMO
The process of homologous recombination is heavily dependent on the RecA family of recombinases for repair of DNA double-strand breaks. These recombinases are responsible for identifying homologies and forming heteroduplex DNA between substrate ssDNA and dsDNA templates, activities that are modified by various accessory factors. In this work we describe the biochemical functions of the SsoRal2 recombinase paralog from the crenarchaeon Sulfolobus solfataricus. We found that the SsoRal2 protein is a DNA-independent ATPase that, unlike the other S. solfataricus paralogs, does not bind either ss- or dsDNA. Instead, SsoRal2 alters the ssDNA binding activity of the SsoRadA recombinase in conjunction with another paralog, SsoRal1. In the presence of SsoRal1, SsoRal2 has a modest effect on strand invasion but effectively abrogates strand exchange activity. Taken together, these results indicate that SsoRal2 assists in nucleoprotein filament modulation and control of strand exchange in S. solfataricus.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Arqueais/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Recombinases/metabolismo , Sulfolobus solfataricus/enzimologia , Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/genética , DNA/química , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Ligação Proteica , Recombinases/química , Recombinases/genética , Sulfolobus solfataricus/genéticaRESUMO
Repair of DNA double-strand breaks is a critical function shared by organisms in all three domains of life. The majority of mechanistic understanding of this process has come from characterization of bacterial and eukaryotic proteins, while significantly less is known about analogous activities in the third, archaeal domain. Despite the physical resemblance of archaea to bacteria, archaeal proteins involved in break repair are remarkably similar to those used by eukaryotes. Investigating the function of the archaeal version of these proteins is, in many cases, simpler than working with eukaryotic homologs owing to their robust nature and ease of purification. In this chapter, we describe methods for purification and activity analysis for the RadA recombinase and its paralogs from the hyperthermophilic acidophilic archaeon Sulfolobus solfataricus.
Assuntos
Proteínas Arqueais/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ensaios Enzimáticos/métodos , Reparo de DNA por Recombinação , Sulfolobus solfataricus/genética , Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrofotometria/instrumentação , Espectrofotometria/métodos , Sulfolobus solfataricus/metabolismoRESUMO
Recombinases of the RecA family play vital roles in homologous recombination, a high-fidelity mechanism to repair DNA double-stranded breaks. These proteins catalyze strand invasion and exchange after forming dynamic nucleoprotein filaments on ssDNA. Increasing evidence suggests that stabilization of these dynamic filaments is a highly conserved function across diverse species. Here, we analyze the presynaptic filament formation and DNA binding characteristics of the Sulfolobus solfataricus recombinase SsoRadA in conjunction with the SsoRadA paralog SsoRal1. In addition to constraining SsoRadA ssDNA-dependent ATPase activity, the paralog also enhances SsoRadA ssDNA binding, effectively influencing activities necessary for presynaptic filament formation. These activities result in enhanced SsoRadA-mediated strand invasion in the presence of SsoRal1 and suggest a filament stabilization function for the SsoRal1 protein.
Assuntos
Proteínas Arqueais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleoproteínas/metabolismo , Sulfolobus solfataricus/enzimologia , Adenosina Trifosfatases/metabolismo , Proteínas Arqueais/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Desoxirribonucleoproteínas/química , Mutação , Ligação Proteica , Sulfolobus solfataricus/metabolismoRESUMO
The mechanisms used by members of the archaeal branch of life to repair DNA damage are not well understood. DNA damage responses have been of particular interest in hyperthermophilic archaea, since these microbes live under environmental conditions that constantly elevate the potential for DNA damage. The work described here focuses on the response of four Sulfolobus solfataricus strains to ionizing radiation (IR) damage. Cellular survival of three wild-type strains and a defined deletion mutant strain was examined following exposure to various IR doses. Using pulsed-field gel electrophoresis, we determined chromosomal DNA double-strand break persistence and repair rates. Among the strains, variable responses were observed, the most surprising of which occurred with the defined deletion mutant strain. This strain displayed higher chromosomal repair rates than the parent strain and was also found to have increased resistance to IR. Using quantitative real-time PCR, we found that transcript levels of homologous recombination-related genes were strongly upregulated following damage in all the strains. The mutant strain again had an enhanced response and dramatically upregulated expression of recombination genes above levels observed for the parent strain, suggesting that increased levels of recombinational repair could account for its increased radiation resistance phenotype. Our results demonstrate a transcriptional response to IR in S. solfataricus for the first time and describe a defined deletion mutant strain that may give the first insight into a damage-based archaeal control element.
Assuntos
Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA , Recombinação Homóloga/efeitos da radiação , Sulfolobus solfataricus/genética , Sulfolobus solfataricus/efeitos da radiação , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Eletroforese em Gel de Campo Pulsado/métodos , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Deleção de Sequência/genética , Deleção de Sequência/efeitos da radiação , Regulação para Cima/efeitos da radiaçãoRESUMO
DNA damage repair mechanisms have been most thoroughly explored in the eubacterial and eukaryotic branches of life. The methods by which members of the archaeal branch repair DNA are significantly less well understood but have been gaining increasing attention. In particular, the approaches employed by hyperthermophilic archaea have been a general source of interest, since these organisms thrive under conditions that likely lead to constant chromosomal damage. In this work we have characterized the responses of three Sulfolobus solfataricus strains to UV-C irradiation, which often results in double-strand break formation. We examined S. solfataricus strain P2 obtained from two different sources and S. solfataricus strain 98/2, a popular strain for site-directed mutation by homologous recombination. Cellular recovery, as determined by survival curves and the ability to return to growth after irradiation, was found to be strain specific and differed depending on the dose applied. Chromosomal damage was directly visualized using pulsed-field gel electrophoresis and demonstrated repair rate variations among the strains following UV-C irradiation-induced double-strand breaks. Several genes involved in double-strand break repair were found to be significantly upregulated after UV-C irradiation. Transcript abundance levels and temporal expression patterns for double-strand break repair genes were also distinct for each strain, indicating that these Sulfolobus solfataricus strains have differential responses to UV-C-induced DNA double-strand break damage.
Assuntos
Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/fisiologia , Sulfolobus solfataricus/genética , Sulfolobus solfataricus/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/efeitos da radiação , Reparo do DNA/genética , Eletroforese em Gel de Campo Pulsado , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In Saccharomyces cerevisiae, the DNA damage response (DDR) is activated by the spatio-temporal colocalization of Mec1-Ddc2 kinase and the 9-1-1 clamp. In the absence of direct means to monitor Mec1 kinase activation in vivo, activation of the checkpoint kinase Rad53 has been taken as a proxy for DDR activation. Here, we identify serine 378 of the Rad55 recombination protein as a direct target site of Mec1. Rad55-S378 phosphorylation leads to an electrophoretic mobility shift of the protein and acts as a sentinel for Mec1 activation in vivo. A single double-stranded break (DSB) in G1-arrested cells causes phosphorylation of Rad55-S378, indicating activation of Mec1 kinase. However, Rad53 kinase is not detectably activated under these conditions. This response required Mec1-Ddc2 and loading of the 9-1-1 clamp by Rad24-RFC, but not Rad9 or Mrc1. In addition to Rad55-S378, two additional direct Mec1 kinase targets are phosphorylated, the middle subunit of the ssDNA-binding protein RPA, RPA2 and histone H2A (H2AX). These data suggest the existence of a truncated signaling pathway in response to a single DSB in G1-arrested cells that activates Mec1 without eliciting a full DDR involving the entire signaling pathway including the effector kinases.
Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Fase G1 , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2 , Reparo do DNA , Proteínas de Ligação a DNA/química , Ensaio de Desvio de Mobilidade Eletroforética , Fase G1/genética , Histonas/metabolismo , Fosforilação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Serina/metabolismoRESUMO
Homologous recombination is an important pathway in the repair of DNA double-strand breaks in all organisms. In mesophiles, single-stranded DNA binding proteins (SSBs) are believed to be involved in the removal of single-stranded DNA (ssDNA) secondary structure during the presynaptic step of homologous recombination, facilitating the formation of a contiguous Rad51/RecA nucleoprotein filament. Here we report a role for the thermophilic archaeal Sulfolobus solfataricus SSB (SsoSSB) in the presynaptic step of homologous recombination. We have identified multiple quaternary structural forms of this protein in vivo and examined the activity of SsoSSB with the strand-exchange protein S. solfataricus RadA (SsoRadA). Using gel-shift analysis, we found that the two major forms of SsoSSB have different DNA binding affinities and site sizes. Biochemical examination of the monomeric form of SsoSSB suggests that it has a minor role in presynapsis and may slightly inhibit the ssDNA-dependent ATPase activity of SsoRadA. The tetrameric form of SsoSSB, however, significantly inhibits SsoRadA ssDNA-dependent ATPase activity under both saturating and subsaturating conditions. Order-of-addition experiments indicate that preincubation of tetrameric SsoSSB and SsoRadA prior to reaction initiation with ssDNA relieves the inhibition observed when SsoSSB is added either before or after SsoRadA. In addition, we demonstrate a direct interaction between SsoRadA and SsoSSB using coimmunoprecipitation. Taken together, these results suggest that a direct interaction between SsoSSB and SsoRadA may occur in vivo prior to the formation of the SsoRadA nucleoprotein filament.
Assuntos
Pareamento Cromossômico , Proteínas de Ligação a DNA/metabolismo , Recombinação Genética , Sulfolobus solfataricus/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Anticorpos/farmacologia , Proteínas Arqueais/antagonistas & inibidores , Cromatografia em Gel , Pareamento Cromossômico/efeitos dos fármacos , Reações Cruzadas/efeitos dos fármacos , Reagentes de Ligações Cruzadas/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Escherichia coli/metabolismo , Fluorescência , Imunoprecipitação , Modelos Biológicos , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Multimerização Proteica/efeitos dos fármacos , Recombinação Genética/efeitos dos fármacos , Sulfolobus solfataricus/efeitos dos fármacos , Sulfolobus solfataricus/enzimologia , TemperaturaRESUMO
DNA damage checkpoints coordinate the cellular response to genotoxic stress and arrest the cell cycle in response to DNA damage and replication fork stalling. Homologous recombination is a ubiquitous pathway for the repair of DNA double-stranded breaks and other checkpoint-inducing lesions. Moreover, homologous recombination is involved in postreplicative tolerance of DNA damage and the recovery of DNA replication after replication fork stalling. Here, we show that the phosphorylation on serines 2, 8, and 14 (S2,8,14) of the Rad55 protein is specifically required for survival as well as for normal growth under genome-wide genotoxic stress. Rad55 is a Rad51 paralog in Saccharomyces cerevisiae and functions in the assembly of the Rad51 filament, a central intermediate in recombinational DNA repair. Phosphorylation-defective rad55-S2,8,14A mutants display a very slow traversal of S phase under DNA-damaging conditions, which is likely due to the slower recovery of stalled replication forks or the slower repair of replication-associated DNA damage. These results suggest that Rad55-S2,8,14 phosphorylation activates recombinational repair, allowing for faster recovery after genotoxic stress.
Assuntos
Dano ao DNA , DNA Fúngico , Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases , Sequência de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2 , Reparo do DNA , Enzimas Reparadoras do DNA , Replicação do DNA , Proteínas de Ligação a DNA/genética , Genoma , Espectrometria de Massas , Modelos Genéticos , Dados de Sequência Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , SerinaRESUMO
Homologous recombination (HR) is a ubiquitous cellular pathway that mediates transfer of genetic information between homologous or near homologous (homeologous) DNA sequences. During meiosis it ensures proper chromosome segregation in the first division. Moreover, HR is critical for the tolerance and repair of DNA damage, as well as in the recovery of stalled and broken replication forks. Together these functions preserve genomic stability and assure high fidelity transmission of the genetic material in the mitotic and meiotic cell divisions. This review will focus on the Rad54 protein, a member of the Snf2-family of SF2 helicases, which translocates on dsDNA but does not display strand displacement activity typical for a helicase. A wealth of genetic, cytological, biochemical and structural data suggests that Rad54 is a core factor of HR, possibly acting at multiple stages during HR in concert with the central homologous pairing protein Rad51.
Assuntos
Recombinação Genética/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases , DNA Helicases , Reparo do DNA/fisiologia , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Modelos Biológicos , Proteína Rad52 de Recombinação e Reparo de DNA/química , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Fatores de Transcrição/fisiologiaRESUMO
The RAD54 gene of Saccharomyces cerevisiae encodes a conserved dsDNA-dependent ATPase of the Swi2/Snf2 family with a specialized function during recombinational DNA repair. Here we analyzed the consequences of the loss of Rad54 function in vegetative (mitotic) cells. Mutants in RAD54 exhibited drastically reduced rates of spontaneous intragenic recombination but were proficient for spontaneous intergenic recombinant formation. The intergenic recombinants likely arose by a RAD54-independent pathway of break-induced replication. Significantly increased rates of spontaneous chromosome loss for diploid rad54/rad54 cells were identified in several independent assays. Inter estingly, the increase in chromosome loss appeared to depend on the presence of a homolog. In addition, the rate of complex genetic events involving chromosome loss were drastically increased in diploid rad54/rad54 cells. Together, these data suggest a role for Rad54 protein in the repair of spontaneous damage, where in the absence of Rad54 protein, homologous recombination is initiated but not properly terminated, leading to misrepair and chromosome loss.
Assuntos
Deleção Cromossômica , Cromossomos Fúngicos , Reparo do DNA , Proteínas Fúngicas/genética , Recombinação Genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , DNA Helicases , Enzimas Reparadoras do DNA , Proteínas Fúngicas/fisiologia , Conversão Gênica , Marcação de Genes , Genoma Fúngico , Histonas/genética , Mitose , Modelos Genéticos , Mutação , Saccharomyces cerevisiae/efeitos da radiação , Raios UltravioletaRESUMO
Trinucleotide repeats (TNRs) undergo frequent mutations in families affected by TNR diseases and in model organisms. Much of the instability is conferred in cis by the sequence and length of the triplet tract. Trans-acting factors also modulate TNR instability risk, on the basis of such evidence as parent-of-origin effects. To help identify trans-acting modifiers, a screen was performed to find yeast mutants with altered CTG.CAG repeat mutation frequencies. The RTG2 gene was identified as one such modifier. In rtg2 mutants, expansions of CTG.CAG repeats show a modest increase in rate, depending on the starting tract length. Surprisingly, contractions were suppressed in an rtg2 background. This creates a situation in a model system where expansions outnumber contractions, as in humans. The rtg2 phenotype was apparently specific for CTG.CAG repeat instability, since no changes in mutation rate were observed for dinucleotide repeats or at the CAN1 reporter gene. This feature sets rtg2 mutants apart from most other mutants that affect genetic stability both for TNRs and at other DNA sequences. It was also found that RTG2 acts independently of its normal partners RTG1 and RTG3, suggesting a novel function of RTG2 that helps modify CTG.CAG repeat mutation risk.