RESUMO
The unique translocation and DNA unwinding properties of DNA helicases can be concealed by the stochastic behavior of enzyme molecules within the necessarily large populations used in ensemble experiments. With recent technological advances, the direct visualization of helicases acting on individual DNA molecules has contributed significantly to the current understanding of their mechanisms of action and biological functions. The combination of single-molecule techniques that enable both manipulation of individual protein or DNA molecules and visualization of their actions has made it possible to literally see novel and unique biochemical characteristics that were previously masked. Here, we describe the execution and use of single-molecule fluorescence imaging techniques, focusing on methods that include optical trapping in conjunction with epifluorescent imaging, and also surface immobilization in conjunction with total internal reflection fluorescence visualization. Combined with microchannel flow cells and microfluidic control, these methods allow individual fluorescently labeled protein and DNA molecules to be imaged and tracked, affording measurement of DNA unwinding and translocation at single-molecule resolution.
Assuntos
Bioquímica/métodos , DNA Helicases/isolamento & purificação , Imagem Molecular/métodos , DNA Helicases/genética , Microscopia de Fluorescência , Pinças ÓpticasRESUMO
The Escherichia coli RecA protein has been a model for understanding homologous eukaryotic recombination proteins such as Rad51. The active form of both RecA and Rad51 appear to be helical filaments polymerized on DNA, in which an unusual helical structure is induced in the DNA. Surprisingly, the human meiosis-specific homolog of RecA, Dmc1, has thus far only been observed to bind DNA as an octameric ring. Sequence analysis and biochemical studies have shown that archaeal RadA proteins are more closely related to Rad51 and Dmc1 than the bacterial RecA proteins. We find that the Sulfolobus solfataricus RadA protein binds DNA in the absence of nucleotide cofactor as an octameric ring and in the presence of ATP as a helical filament. Since it is likely that RadA is closely related to a common ancestral protein of both Rad51 and Dmc1, the two DNA-binding forms of RadA may provide insight into the divergence that has taken place between Rad51 and Dmc1.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Proteínas de Ciclo Celular , DNA Arqueal/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , DNA Arqueal/genética , Humanos , Microscopia Eletrônica , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Rad51 RecombinaseRESUMO
In the October 5 issue of Cell, Singleton et al. report the crystal structure of RecG protein bound to an analog of a stalled DNA replication fork. This structure shows how RecG can recognize branched DNA structures and suggests a mechanism for fork reversal, an early event in recombination-dependent reinitiation of DNA replication.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Helicases/metabolismo , Replicação do DNA , Proteínas de Escherichia coli , Proteínas de Bactérias/química , DNA Helicases/química , Escherichia coli/genética , Modelos Biológicos , Conformação de Ácido NucleicoRESUMO
RAD54 is an important member of the RAD52 group of genes that carry out recombinational repair of DNA damage in the yeast Saccharomyces cerevisiae. Rad54 protein is a member of the Snf2/Swi2 protein family of DNA-dependent/stimulated ATPases, and its ATPase activity is crucial for Rad54 protein function. Rad54 protein and Rad54-K341R, a mutant protein defective in the Walker A box ATP-binding fold, were fused to glutathione-S-transferase (GST) and purified to near homogeneity. In vivo, GST-Rad54 protein carried out the functions required for methyl methanesulfonate sulfate (MMS), UV, and DSB repair. In vitro, GST-Rad54 protein exhibited dsDNA-specific ATPase activity. Rad54 protein stimulated Rad51/Rpa-mediated DNA strand exchange by specifically increasing the kinetics of joint molecule formation. This stimulation was accompanied by a concurrent increase in the formation of heteroduplex DNA. Our results suggest that Rad54 protein interacts specifically with established Rad51 nucleoprotein filaments before homology search on the duplex DNA and heteroduplex DNA formation. Rad54 protein did not stimulate DNA strand exchange by increasing presynaptic complex formation. We conclude that Rad54 protein acts during the synaptic phase of DNA strand exchange and after the formation of presynaptic Rad51 protein-ssDNA filaments.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Fúngicas/metabolismo , Ácidos Nucleicos Heteroduplexes/metabolismo , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Pareamento de Bases , DNA/química , DNA/genética , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , DNA Helicases , Reparo do DNA/genética , Enzimas Reparadoras do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Teste de Complementação Genética , Cinética , Metanossulfonato de Metila/farmacologia , Modelos Genéticos , Mutação/genética , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/genética , Rad51 Recombinase , Recombinases Rec A/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Temperatura , Raios UltravioletaRESUMO
RecBCD enzyme is a processive DNA helicase and nuclease that participates in the repair of chromosomal DNA through homologous recombination. We have visualized directly the movement of individual RecBCD enzymes on single molecules of double-stranded DNA (dsDNA). Detection involves the optical trapping of solitary, fluorescently tagged dsDNA molecules that are attached to polystyrene beads, and their visualization by fluorescence microscopy. Both helicase translocation and DNA unwinding are monitored by the displacement of fluorescent dye from the DNA by the enzyme. Here we show that unwinding is both continuous and processive, occurring at a maximum rate of 972 +/- 172 base pairs per second (0.30 microm s(-1)), with as many as 42,300 base pairs of dsDNA unwound by a single RecBCD enzyme molecule. The mean behaviour of the individual RecBCD enzyme molecules corresponds to that observed in bulk solution.
Assuntos
DNA Helicases/metabolismo , DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Trifosfato de Adenosina/metabolismo , Transporte Biológico , DNA Viral , Exodesoxirribonuclease V , Processamento de Imagem Assistida por Computador , Lasers , Microscopia de Fluorescência , Microscopia de Vídeo , Óptica e FotônicaRESUMO
We demonstrate that RecQ helicase from Escherichia coli is a catalytic helicase whose activity depends on the concentration of ATP, free magnesium ion, and single-stranded DNA-binding (SSB) protein. Helicase activity is cooperative in ATP concentration, with an apparent S(0.5) value for ATP of 200 microm and a Hill coefficient of 3.3 +/- 0.3. Therefore, RecQ helicase utilizes multiple, interacting ATP-binding sites to mediate double-stranded DNA (dsDNA) unwinding, implicating a multimer of at least three subunits as the active unwinding species. Unwinding activity is independent of dsDNA ends, indicating that RecQ helicase can unwind from both internal regions and ends of dsDNA. The K(M) for dsDNA is 0.5-0.9 microm base pairs; the k(cat) for DNA unwinding is 2.3-2.7 base pairs/s/monomer of RecQ helicase; and unexpectedly, helicase activity is optimal at a free magnesium ion concentration of 0.05 mm. Omitting Escherichia coli SSB protein lowers the rate and extent of dsDNA unwinding, suggesting that RecQ helicase associates with the single-stranded DNA (ssDNA) product. In agreement, the ssDNA-dependent ATPase activity is reduced in proportion to the SSB protein concentration; in its absence, ATPase activity saturates at six nucleotides/RecQ helicase monomer and yields a k(cat) of 24 s(-1). Thus, we conclude that SSB protein stimulates RecQ helicase-mediated unwinding by both trapping the separated ssDNA strands after unwinding and preventing the formation of non-productive enzyme-ssDNA complexes.
Assuntos
Adenosina Trifosfatases/metabolismo , DNA Helicases/metabolismo , DNA/metabolismo , Escherichia coli/enzimologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Bisbenzimidazol/metabolismo , DNA/química , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Escherichia coli/metabolismo , Corantes Fluorescentes/metabolismo , Cinética , Magnésio/farmacologia , Modelos Biológicos , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , RecQ Helicases , Especificidade por SubstratoRESUMO
Rad51 and Rad54 proteins are important for the repair of double-stranded DNA (dsDNA) breaks by homologous recombination in eukaryotes. Rad51 assembles on single-stranded DNA (ssDNA) to form a helical nucleoprotein filament that performs homologous pairing with dsDNA; Rad54 stimulates this pairing substantially. Here, we demonstrate that Rad54 acts in concert with the mature Rad51-ssDNA filament. Enhancement of DNA pairing by Rad54 is greatest at an equimolar ratio relative to Rad51 within the filament. Reciprocally, the Rad51-ssDNA filament enhances both the dsDNA-dependent ATPase and the dsDNA unwinding activities of Rad54. We conclude that Rad54 participates in the DNA homology search as a component of the Rad51-nucleoprotein filament and that the filament delivers Rad54 to the dsDNA pairing locus, thereby linking the unwinding of potential target DNA with the homology search process.
Assuntos
Reparo do DNA/fisiologia , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatases/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , DNA Helicases , Reparo do DNA/efeitos dos fármacos , Enzimas Reparadoras do DNA , DNA Super-Helicoidal/metabolismo , Proteínas de Ligação a DNA/farmacologia , Escherichia coli , Proteínas Fúngicas/farmacologia , Humanos , Rad51 Recombinase , Saccharomyces cerevisiae , Especificidade da EspécieRESUMO
The archaeal RadA protein is a homologue of the Escherichia coli RecA and Saccharomyces cerevisiae Rad51 proteins and possesses the same biochemical activities. Here, using in vitro selection, we show that the Sulfolobus solfataricus RadA protein displays the same preference as its homologues for binding to DNA sequences that are rich in G residues, and under-represented in A and C residues. The RadA protein also displays enhanced pairing activity with these in vitro-selected sequences. These parallels between the archaeal, eukaryal and bacterial proteins further extend the universal characteristics of DNA strand exchange proteins.
Assuntos
Proteínas Arqueais/metabolismo , DNA Arqueal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli , Sulfolobus/metabolismo , Proteínas Arqueais/genética , DNA Arqueal/genética , Proteínas de Ligação a DNA/genética , Ligação Proteica , Sulfolobus/genéticaRESUMO
In wild-type Escherichia coli, recognition of the recombination hotspot, chi (5'-GCTGGTGG-3'), by the RecBCD enzyme is central to homologous recombination. However, in the recC* class of RecBCD mutants, stimulation of recombination by the canonical chi sequence is not detectable, but the levels of homologous recombination are nearly wild-type. In vivo studies demonstrate that a member of this class of mutants, the recC1004 allele, encodes an enzyme that responds to a novel variant of chi, termed chi* (5'-GCTGGTGCTCG-3'). Here, we establish that, in vitro, the chi* sequence is recognized more efficiently by the RecBC(1004)D enzyme than is the wild-type chi. This is manifest by both a greater modification of nuclease activity and a higher stimulation of RecA protein-mediated joint molecule formation at chi* than at chi. Sequencing of the recC1004 gene revealed that it contains a frameshift mutation, which results in a replacement of nine of the wild-type amino acid residues by eight in the mutant protein, and defines a locus that is important for the specificity of chi-recognition. In addition, we show that this novel, 11 nucleotide chi* sequence also regulates the wild-type RecBCD enzyme, supporting the notion that variants of the canonical chi constitute a class of sequences that regulate the recombination function of RecBCD enzyme.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/enzimologia , Escherichia coli/genética , Exodesoxirribonucleases/metabolismo , Mutação/genética , Recombinação Genética/genética , Sequências Reguladoras de Ácido Nucleico/genética , Alelos , Sequência de Aminoácidos , Pareamento de Bases/genética , Sequência de Bases , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Ativação Enzimática , Exodesoxirribonuclease V , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Variação Genética/genética , Dados de Sequência Molecular , Nucleotídeos/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Recombinases Rec A/metabolismo , Especificidade por SubstratoRESUMO
DNA helicases are ubiquitous enzymes that unwind double-stranded DNA. They are a diverse group of proteins that move in a linear fashion along a one-dimensional polymer lattice--DNA--by using a mechanism that couples nucleoside triphosphate hydrolysis to both translocation and double-stranded DNA unwinding to produce separate strands of DNA. The RecBC enzyme is a processive DNA helicase that functions in homologous recombination in Escherichia coli; it unwinds up to 6,250 base pairs per binding event and hydrolyses slightly more than one ATP molecule per base pair unwound. Here we show, by using a series of gapped oligonucleotide substrates, that this enzyme translocates along only one strand of duplex DNA in the 3'-->5' direction. The translocating enzyme will traverse, or 'step' across, single-stranded DNA gaps in defined steps that are 23 (+/-2) nucleotides in length. This step is much larger than the amount of double-stranded DNA that can be unwound using the free energy derived from hydrolysis of one molecule of ATP, implying that translocation and DNA unwinding are separate events. We propose that the RecBC enzyme both translocates and unwinds by a quantized, two-step, inchworm-like mechanism that may have parallels for translocation by other linear motor proteins.
Assuntos
DNA Helicases/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Exodesoxirribonucleases/metabolismo , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Exodesoxirribonuclease V , Heparina/metabolismo , Especificidade por SubstratoRESUMO
Although the RecB(2109)CD enzyme retains most of the biochemical functions associated with the wild-type RecBCD enzyme, it is completely defective for genetic recombination. Here, we demonstrate that the mutant enzyme exhibits an aberrant double-stranded DNA exonuclease activity, intrinsically producing a 3'-terminal single-stranded DNA overhang that is an ideal substrate for RecA protein-promoted strand invasion. Thus, the mutant enzyme constitutively processes double-stranded DNA in the same manner as the chi-modified wild-type RecBCD enzyme. However, we further show that the RecB(2109)CD enzyme is unable to coordinate the loading of RecA protein onto the single-stranded DNA produced, and we conclude that this inability results in the recombination-defective phenotype of the recB2109 allele. Our findings argue that the facilitated loading of RecA protein by the chi-activated RecBCD enzyme is essential for RecBCD-mediated homologous recombination in vivo.
Assuntos
Proteínas de Escherichia coli , Exodesoxirribonucleases/genética , Recombinases Rec A/metabolismo , Recombinação Genética , DNA de Cadeia Simples/metabolismo , Eletroforese em Gel de Poliacrilamida , Exodesoxirribonuclease V , Exodesoxirribonucleases/metabolismo , Modelos Químicos , Dados de Sequência Molecular , Mutagênese , FenótipoRESUMO
Recombination initiates at double-stranded DNA breaks and at single-stranded DNA gaps. These DNA strand discontinuities can arise from DNA-damaging agents and from normal DNA replication when the DNA polymerase encounters an imperfection in the DNA template or another protein. The machinery of homologous recombination acts at these breaks and gaps to promote the events that result in gene recombination, as well as the reattachment of detached replication arms and the resumption of DNA replication. In Escherichia coli, these events require collaboration (RecA, RecBCD, RecFOR, RecQ, RuvABC and SSB proteins) and DNA replication (PriABC proteins and the DNA polymerases). The initial steps common to these recombination and recombination-dependent replication processes are reviewed.
Assuntos
Replicação do DNA/genética , Recombinação Genética , Dano ao DNA , Escherichia coli/genéticaRESUMO
The AddAB enzyme is important to homologous DNA recombination in Bacillus subtilis, where it is thought to be the functional counterpart of the RecBCD enzyme of Escherichia coli. In vivo, AddAB responds to a specific five-nucleotide sequence (5'-AGCGG-3' or its complement) in a manner analogous to the response of the RecBCD enzyme to interaction with chi sequences. Here, we show that purified AddAB enzyme is able to load at a double-stranded DNA end and is both a DNA helicase and nuclease, whose combined action results in the degradation of both strands of the DNA duplex. During translocation, recognition of the properly oriented sequence 5'-AGCGG-3' causes attenuation of the AddAB enzyme nuclease activity that is responsible for degradation of the strand 3'-terminal at the entry site. Therefore, we conclude that 5'-AGCGG-3' is the B. subtilis Chi site and it is hereafter referred to as chi(Bs). After encountering chi(Bs), both the degradation of the 5'-terminal strand and the helicase activity persist. Thus, processing of a double-stranded DNA end by the AddAB enzyme produces a duplex DNA molecule with a protruding 3'-terminated single-stranded tail, a universal intermediate of the recombination process.
Assuntos
Sítios de Ligação Microbiológicos/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , DNA Helicases/metabolismo , Exodesoxirribonucleases/metabolismo , Exonucleases/metabolismo , Recombinação Genética/genética , Sequências Reguladoras de Ácido Nucleico/genética , Sequência de Bases , DNA Helicases/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Exodesoxirribonuclease V , Exodesoxirribonucleases/química , Exonucleases/química , Magnésio/metabolismo , Peso Molecular , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Genetic recombination in Escherichia coli is stimulated by the recombination hotspot Chi (chi), a regulatory element that modifies the activities of the RecBCD enzyme and leads to loading of the DNA strand exchange protein, RecA, onto the chi-containing DNA strand. The RecBC enzyme, which lacks the RecD subunit, loads RecA protein constitutively, in a manner that is independent of chi. Using a truncated RecBC enzyme lacking the 30 kDa C-terminal domain of the RecB subunit, we show that this domain is necessary for RecA protein-loading. We propose that this domain harbors a site that interacts with RecA protein, recruiting it to single-stranded DNA during unwinding. This ability of a translocating enzyme to deliver material (RecA protein) to a specific target site (the chi sequence) parallels that of other cellular motor proteins.
Assuntos
DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Recombinases Rec A/metabolismo , Pareamento de Bases/genética , DNA Helicases/química , DNA Helicases/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA Super-Helicoidal/química , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonuclease V , Exodesoxirribonucleases/genética , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Modelos Biológicos , Ligação Proteica , Recombinação Genética/genética , Sequências Reguladoras de Ácido Nucleico/genética , Deleção de Sequência/genéticaRESUMO
Traditionally, recombination reactions promoted by RecA-like proteins initiate by forming a nucleoprotein filament on a single-stranded DNA (ssDNA), which then pairs with homologous double-stranded DNA (dsDNA). In this paper, we describe a novel pairing process that occurs in an unconventional manner: RecA protein polymerizes along dsDNA to form an active nucleoprotein filament that can pair and exchange strands with homologous ssDNA. Our results demonstrate that this "inverse" reaction is a unique, highly efficient DNA strand exchange reaction that is not due to redistribution of RecA protein from dsDNA to the homologous ssDNA partner. Finally, we demonstrate that the RecA protein-dsDNA filament can also pair and promote strand exchange with ssRNA. This inverse RNA strand exchange reaction is likely responsible for R-loop formation that is required for recombination-dependent DNA replication.
Assuntos
DNA Bacteriano/genética , Escherichia coli/metabolismo , RNA Bacteriano/genética , Recombinases Rec A/metabolismo , Sequência de Bases , Escherichia coli/genética , Dados de Sequência MolecularRESUMO
The repair of potentially lethal DNA double-stranded breaks (DSBs) by homologous recombination requires processing of the broken DNA into a resected DNA duplex with a protruding 3'-single-stranded DNA (ssDNA) tail. Accordingly, the canonical models for DSB repair require invasion of an intact homologous DNA template by the 3'-end of the ssDNA, a characteristic that the bacterial pairing protein RecA possesses. Unexpectedly, we find that for the eukaryotic homolog, Rad51 protein, the 5'-end of ssDNA is more invasive than the 3'-end. This pairing bias is unaffected by Rad52, Rad54 or Rad55-57 proteins. However, further investigation reveals that, in contrast to RecA protein, the preferred DNA substrate for Rad51 protein is not ssDNA but rather dsDNA with ssDNA tails. This important distinction permits the Rad51 proteins to promote DNA strand invasion using either 3'- or 5'-ends with similar efficiency.
Assuntos
Pareamento de Bases , Proteínas de Ligação a DNA/genética , DNA/genética , Recombinação Genética , Reparo do DNA , Escherichia coli , Humanos , Rad51 Recombinase , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiaeRESUMO
The RecA142 protein, in which valine is substituted for isoleucine-225, is defective for genetic recombination in vivo and for DNA strand exchange activity in vitro under conventional growth and reaction conditions respectively. However, we show that mildly acidic conditions restore both the in vitro DNA strand exchange activity and the in vivo function of RecA142 protein, suggesting that recombination function can be restored by a slight change in protein structure elicited by protonation. Indeed, we identified an intragenic suppressor of the recombination deficiency of the recA142 allele. This suppressor mutation is a substitution of leucine for glutamine at position 124. Based on the three-dimensional structure, the Q-124L substitution is predicted to make a new monomer-monomer contact with residue phenylalanine-21 of the adjacent RecA monomer. The Q-124L mutation is not allele specific, because it also suppresses the recombination deficiency of a recA deletion (Delta9), lacking nine amino acids at the amino-terminus, presumably by reinforcing the monomer-monomer interactions that are attenuated by the Delta9 deletion. Expression of RecA(Q-124L) protein is toxic to Escherichia coli, presumably because of enhanced affinity for DNA. We speculate as to how enhanced monomer-monomer interactions and acidic pH conditions can restore the recombination activity of some defective recA alleles.