RESUMO
Tethering beads to DNA offers a panel of single molecule techniques for the refined analysis of the conformational dynamics of DNA and the elucidation of the mechanisms of enzyme activity. Recent developments include the massive parallelization of these techniques achieved by the fabrication of dedicated nanoarrays by soft nanolithography. We focus here on two of these techniques: the Tethered Particle motion and Magnetic Tweezers allowing analysis of the behavior of individual DNA molecules in the absence of force and under the application of a force and/or a torque, respectively. We introduce the experimental protocols for the parallelization and discuss the benefits already gained, and to come, for these single molecule investigations.
Assuntos
DNA/química , Pinças Ópticas , Imagem Individual de Molécula/métodos , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Magnetismo/métodos , Movimento (Física) , Nanotecnologia/métodos , Conformação de Ácido NucleicoRESUMO
Ribonucleotides are the natural analogs of deoxyribonucleotides, which can be misinserted by DNA polymerases, leading to the most abundant DNA lesions in genomes. During replication, DNA polymerases tolerate patches of ribonucleotides on the parental strands to different extents. The majority of human DNA polymerases have been reported to misinsert ribonucleotides into genomes. However, only PrimPol, DNA polymerase α, telomerase, and the mitochondrial human DNA polymerase (hpol) γ have been shown to tolerate an entire RNA strand. Y-family hpol η is known for translesion synthesis opposite the UV-induced DNA lesion cyclobutane pyrimidine dimer and was recently found to incorporate ribonucleotides into DNA. Here, we report that hpol η is able to bind DNA/DNA, RNA/DNA, and DNA/RNA duplexes with similar affinities. In addition, hpol η, as well as another Y-family DNA polymerase, hpol κ, accommodates RNA as one of the two strands during primer extension, mainly by inserting dNMPs opposite unmodified templates or DNA lesions, such as 8-oxo-2'-deoxyguanosine or cyclobutane pyrimidine dimer, even in the presence of an equal amount of the DNA/DNA substrate. The discovery of this RNA-accommodating ability of hpol η redefines the traditional concept of human DNA polymerases and indicates potential new functions of hpol η in vivo.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , RNA/metabolismo , Elongação da Transcrição Genética , 8-Hidroxi-2'-Desoxiguanosina , Pareamento Incorreto de Bases , Primers do DNA/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/genética , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Cinética , Ácidos Nucleicos Heteroduplexes , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/metabolismo , Oligorribonucleotídeos/metabolismo , Dímeros de Pirimidina/metabolismo , Proteínas Recombinantes/metabolismo , Transcrição Reversa , Especificidade por SubstratoRESUMO
MukB is a structural maintenance of chromosome-like protein required for DNA condensation. The complete condensin is a large tripartite complex of MukB, the kleisin, MukF, and an accessory protein, MukE. As found previously, MukB DNA condensation is a stepwise process. We have defined these steps topologically. They proceed first via the formation of negative supercoils that are sequestered by the protein followed by hinge-hinge interactions between MukB dimers that stabilize topologically isolated loops in the DNA. MukB itself is sufficient to mediate both of these topological alterations; neither ATP nor MukEF is required. We show that the MukB hinge region binds DNA and that this region of the protein is involved in sequestration of supercoils. Cells carrying mutations in the MukB hinge that reduce DNA condensation in vitro exhibit nucleoid decondensation in vivo.
Assuntos
Proteínas Cromossômicas não Histona/química , DNA Bacteriano/química , DNA Super-Helicoidal/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Multimerização Proteica , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Properly condensed chromosomes are necessary for accurate segregation of the sisters after DNA replication. The Escherichia coli condesin is MukB, a structural maintenance of chromosomes (SMC)-like protein, which forms a complex with MukE and the kleisin MukF. MukB is known to be able to mediate knotting of a DNA ring, an intramolecular reaction. In our investigations of how MukB condenses DNA we discovered that it can also mediate catenation of two DNA rings, an intermolecular reaction. This activity of MukB requires DNA binding by the head domains of the protein but does not require either ATP or its partner proteins MukE or MukF. The ability of MukB to mediate DNA catenation underscores its potential for bringing distal regions of a chromosome together.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , DNA Bacteriano/metabolismo , DNA Catenado/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Repressoras/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Catenado/química , DNA Catenado/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas Repressoras/química , Proteínas Repressoras/genéticaRESUMO
Reverse gyrase is a DNA topoisomerase specific for hyperthermophilic bacteria and archaea. It catalyzes the peculiar ATP-dependent DNA-positive supercoiling reaction and might be involved in the physiological adaptation to high growth temperature. Reverse gyrase comprises an N-terminal ATPase and a C-terminal topoisomerase domain, which cooperate in enzyme activity, but details of its mechanism of action are still not clear. We present here a functional characterization of PcalRG, a novel reverse gyrase from the archaeon Pyrobaculum calidifontis. PcalRG is the most robust and processive reverse gyrase known to date; it is active over a wide range of conditions, including temperature, ionic strength, and ATP concentration. Moreover, it holds a strong ATP-inhibited DNA cleavage activity. Most important, PcalRG is able to induce ATP-dependent unwinding of synthetic Holliday junctions and ATP-stimulated annealing of unconstrained single-stranded oligonucleotides. Combined DNA unwinding and annealing activities are typical of certain helicases, but until now were shown for no other reverse gyrase. Our results suggest for the first time that a reverse gyrase shares not only structural but also functional features with evolutionary conserved helicase-topoisomerase complexes involved in genome stability.
Assuntos
Proteínas Arqueais/química , DNA Topoisomerases Tipo I/química , DNA Arqueal/química , Pyrobaculum/enzimologia , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Arqueal/genética , DNA Arqueal/metabolismo , Evolução Molecular , Instabilidade Genômica/fisiologia , Pyrobaculum/genéticaRESUMO
Metnase (or SETMAR) arose from a chimeric fusion of the Hsmar1 transposase downstream of a protein methylase in anthropoid primates. Although the Metnase transposase domain has been largely conserved, its catalytic motif (DDN) differs from the DDD motif of related transposases, which may be important for its role as a DNA repair factor and its enzymatic activities. Here, we show that substitution of DDN(610) with either DDD(610) or DDE(610) significantly reduced in vivo functions of Metnase in NHEJ repair and accelerated restart of replication forks. We next tested whether the DDD or DDE mutants cleave single-strand extensions and flaps in partial duplex DNA and pseudo-Tyr structures that mimic stalled replication forks. Neither substrate is cleaved by the DDD or DDE mutant, under the conditions where wild-type Metnase effectively cleaves ssDNA overhangs. We then characterized the ssDNA-binding activity of the Metnase transposase domain and found that the catalytic domain binds ssDNA but not dsDNA, whereas dsDNA binding activity resides in the helix-turn-helix DNA binding domain. Substitution of Asn-610 with either Asp or Glu within the transposase domain significantly reduces ssDNA binding activity. Collectively, our results suggest that a single mutation DDN(610) â DDD(610), which restores the ancestral catalytic site, results in loss of function in Metnase.
Assuntos
Reparo do DNA por Junção de Extremidades , Replicação do DNA , Histona-Lisina N-Metiltransferase/química , Motivos de Aminoácidos , Asparagina/química , Sequência de Bases , Domínio Catalítico , Núcleo Celular/metabolismo , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Histonas/química , Humanos , Dados de Sequência Molecular , Ligação Proteica , Interferência de RNA , Transposases/metabolismoRESUMO
Crossing over between homologous chromosomes is initiated in meiotic prophase in most sexually reproducing organisms by the appearance of programmed double strand breaks throughout the genome. In Saccharomyces cerevisiae the double-strand breaks are resected to form three prime single-strand tails that primarily invade complementary sequences in unbroken homologs. These invasion intermediates are converted into double Holliday junctions and then resolved into crossovers that facilitate homolog segregation during Meiosis I. Work in yeast suggests that Msh4-Msh5 stabilizes invasion intermediates and double Holliday junctions, which are resolved into crossovers in steps requiring Sgs1 helicase, Exo1, and a putative endonuclease activity encoded by the DNA mismatch repair factor Mlh1-Mlh3. We purified Mlh1-Mlh3 and showed that it is a metal-dependent and Msh2-Msh3-stimulated endonuclease that makes single-strand breaks in supercoiled DNA. These observations support a direct role for an Mlh1-Mlh3 endonuclease activity in resolving recombination intermediates and in DNA mismatch repair.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , DNA Cruciforme/metabolismo , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/metabolismo , Meiose/fisiologia , Proteína 2 Homóloga a MutS/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Quebras de DNA de Cadeia Simples , DNA Cruciforme/genética , DNA Fúngico/genética , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/metabolismo , Proteínas de Ligação a DNA/genética , Desoxirribonuclease I/genética , Proteína 1 Homóloga a MutL , Proteínas MutL , Proteína 2 Homóloga a MutS/genética , Proteína 3 Homóloga a MutS , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Inteins are mobile genetic elements capable of self-splicing post-translationally. They exist in all three domains of life including in viruses and bacteriophage, where they have a sporadic distribution even among very closely related species. In this review, we address this anomalous distribution from the point of view of the evolution of the host species as well as the intrinsic features of the inteins that contribute to their genetic mobility. We also discuss the incidence of inteins in functionally important sites of their host proteins. Finally, we describe instances of conditional protein splicing. These latter observations lead us to the hypothesis that some inteins have adapted to become sensors that play regulatory roles within their host protein, to the advantage of the organism in which they reside.
Assuntos
Evolução Molecular , Inteínas/genética , Processamento de Proteína/genética , Proteínas/genética , Sequência de Aminoácidos , Archaea/genética , Bactérias/genética , Eucariotos/genética , Genoma/genética , Modelos GenéticosRESUMO
Clamp loaders belong to a family of proteins known as ATPases associated with various cellular activities (AAA+). These proteins utilize the energy from ATP binding and hydrolysis to perform cellular functions. The clamp loader is required to load the clamp onto DNA for use by DNA polymerases to increase processivity. ATP binding and hydrolysis are coordinated by several key residues, including a conserved Lys located within the Walker A motif (or P-loop). This residue is required for each subunit to bind ATP. The specific function of each ATP molecule bound to the Saccharomyces cerevisiae clamp loader is unknown. A series of point mutants, each lacking a single Walker A Lys residue, was generated to study the effects of abolishing ATP binding in individual clamp loader subunits. A variety of biochemical assays were used to analyze the function of ATP binding during discrete steps of the clamp loading reaction. All mutants reduced clamp binding/opening to different degrees. Decreased clamp binding activity was generally correlated with decreases in the population of open clamps, suggesting that differences in the binding affinities of Walker A mutants stem from differences in stabilization of proliferating cell nuclear antigen in an open conformation. Walker A mutations had a smaller effect on DNA binding than clamp binding/opening. Our data do not support a model in which each ATP site functions independently to regulate a different step in the clamp loading cycle to coordinate these steps. Instead, the ATP sites work in unison to promote conformational changes in the clamp loader that drive clamp loading.
Assuntos
DNA Fúngico/química , DNA Polimerase Dirigida por DNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Motivos de Aminoácidos , DNA Fúngico/biossíntese , DNA Fúngico/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Mutação Puntual , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
3-Methyladenine DNA glycosylase recognizes and excises a wide range of damaged bases and thus plays a critical role in base excision repair. However, knowledge on the regulation of DNA glycosylase in prokaryotes and eukaryotes is limited. In this study, we successfully characterized a TetR family transcriptional factor from Mycobacterium bovis bacillus Calmette-Guerin (BCG), namely BCG0878c, which directly regulates the expression of 3-methyladenine DNA glycosylase (designated as MbAAG) and influences the base excision activity of this glycosylase at the post-translational level. Using electrophoretic mobility shift assay and DNase I footprinting experiments, we identified two conserved motifs within the upstream region of mbaag specifically recognized by BCG0878c. Significant down-regulation of mbaag was observed in BCG0878c-overexpressed M. bovis BCG strains. By contrast, about 12-fold up-regulation of mbaag expression was found in bcg0878c-deleted mutant M. bovis BCG strains. ß-Galactosidase activity assays also confirmed these results. Thus, BCG0878c can function as a negative regulator of mbaag expression. In addition, the regulator was shown to physically interact with MbAAG to enhance the ability of the glycosylase to bind damaged DNA. Interaction between the two proteins was further found to facilitate AAG-catalyzed removal of hypoxanthine from DNA. These results indicate that a TetR family protein can dually regulate the function of 3-methyladenine DNA glycosylase in M. bovis BCG both at the transcriptional and post-translational levels. These findings enhance our understanding of the expression and regulation of AAG in mycobacteria.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Glicosilases/genética , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium bovis/enzimologia , Mycobacterium bovis/genética , Fatores de Transcrição/metabolismo , Sequência de Bases , Dano ao DNA , DNA Bacteriano/metabolismo , Dados de Sequência Molecular , Mycobacterium bovis/metabolismo , Motivos de Nucleotídeos , Ligação ProteicaRESUMO
BACKGROUND: Base excision repair is hindered by nucleosomes. RESULTS: Outwardly oriented uracils near the nucleosome center are efficiently cleaved; however, polymerase ß is strongly inhibited at these sites. CONCLUSION: The histone octamer presents different levels of constraints on BER, dependent on the structural requirements for enzyme activity. SIGNIFICANCE: Chromatin remodeling is necessary to prevent accumulation of aborted intermediates in nucleosomes. Packaging of DNA into chromatin affects accessibility of DNA regulatory factors involved in transcription, replication, and repair. Evidence suggests that even in the nucleosome core particle (NCP), accessibility to damaged DNA is hindered by the presence of the histone octamer. Base excision repair is the major pathway in mammalian cells responsible for correcting a large number of chemically modified bases. We have measured the repair of site-specific uracil and single nucleotide gaps along the surface of the NCP. Our results indicate that removal of DNA lesions is greatly dependent on their rotational and translational positioning in NCPs. Significantly, the rate of uracil removal with outwardly oriented DNA backbones is 2-10-fold higher than those with inwardly oriented backbones. In general, uracils with inwardly oriented backbones farther away from the dyad center of the NCP are more accessible than those near the dyad. The translational positioning of outwardly oriented gaps is the key factor driving gap filling activity. An outwardly oriented gap near the DNA ends exhibits a 3-fold increase in gap filling activity as compared with one near the dyad with the same rotational orientation. Near the dyad, uracil DNA glycosylase/APE1 removes an outwardly oriented uracil efficiently; however, polymerase ß activity is significantly inhibited at this site. These data suggest that the hindrance presented by the location of a DNA lesion is dependent on the structural requirements for enzyme catalysis. Therefore, remodeling at DNA damage sites in NCPs is critical for preventing accumulation of aborted intermediates and ensuring completion of base excision repair.
Assuntos
Dano ao DNA , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Proteínas de Escherichia coli/química , Nucleossomos/química , Uracila-DNA Glicosidase/química , Animais , Ácido Apurínico/química , Galinhas , Reagentes de Ligações Cruzadas/química , DNA/química , DNA Polimerase beta/química , Eritrócitos/química , Formaldeído/química , Humanos , Hidrólise , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Uracila/químicaRESUMO
TREX1 is an autonomous 3'-exonuclease that degrades DNA to prevent inappropriate immune activation. The TREX1 protein is composed of 314 amino acids; the N-terminal 242 amino acids contain the catalytic domain, and the C-terminal region (CTR) localizes TREX1 to the cytosolic compartment. In this study, we show that TREX1 modification by ubiquitination is controlled by a highly conserved sequence in the CTR to affect cellular localization. Transfection of TREX1 deletion constructs into human cells demonstrated that this sequence is required for ubiquitination at multiple lysine residues through a "non-canonical" ubiquitin linkage. A proteomic approach identified ubiquilin 1 as a TREX1 CTR-interacting protein, and this interaction was verified in vitro and in vivo. Cotransfection studies indicated that ubiquilin 1 localizes TREX1 to cytosolic punctate structures dependent upon the TREX1 CTR and lysines within the TREX1 catalytic core. Several TREX1 mutants linked to the autoimmune diseases Aicardi-Goutières syndrome and systemic lupus erythematosus that exhibit full catalytic function were tested for altered ubiquitin modification and cellular localization. Our data show that these catalytically competent disease-causing TREX1 mutants exhibit differential levels of ubiquitination relative to WT TREX1, suggesting a novel mechanism of dysfunction. Furthermore, these differentially ubiquitinated disease-causing mutants also exhibit altered ubiquilin 1 co-localization. Thus, TREX1 post-translational modification indicates an additional mechanism by which mutations disrupt TREX1 biology, leading to human autoimmune disease.
Assuntos
Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Ubiquitinação , Proteínas Adaptadoras de Transdução de Sinal , Doenças Autoimunes do Sistema Nervoso/metabolismo , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células HEK293 , Humanos , Lúpus Eritematoso Sistêmico/metabolismo , Lisina/metabolismo , Proteínas Mutantes/metabolismo , Malformações do Sistema Nervoso/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico , Relação Estrutura-AtividadeRESUMO
Expansion of CAG/CTG trinucleotide repeats causes certain familial neurological disorders. Hairpin formation in the nascent strand during DNA synthesis is considered a major path for CAG/CTG repeat expansion. However, the underlying mechanism is unclear. We show here that removal or retention of a nascent strand hairpin during DNA synthesis depends on hairpin structures and types of DNA polymerases. Polymerase (pol) δ alone removes the 3'-slipped hairpin using its 3'-5' proofreading activity when the hairpin contains no immediate 3' complementary sequences. However, in the presence of pol ß, pol δ preferentially facilitates hairpin retention regardless of hairpin structures. In this reaction, pol ß incorporates several nucleotides to the hairpin 3'-end, which serves as an effective primer for the continuous DNA synthesis by pol δ, thereby leading to hairpin retention and repeat expansion. These findings strongly suggest that coordinated processing of 3'-slipped (CAG)n/(CTG)n hairpins by polymerases δ and ß on during DNA synthesis induces CAG/CTG repeat expansions.
Assuntos
DNA Polimerase III/metabolismo , DNA Polimerase beta/metabolismo , Replicação do DNA/fisiologia , DNA/biossíntese , Sequências Repetidas Invertidas , DNA/química , DNA/genética , DNA Polimerase III/química , DNA Polimerase III/genética , DNA Polimerase beta/química , DNA Polimerase beta/genética , Células HeLa , HumanosRESUMO
The resection of DNA double strand breaks initiates homologous recombination (HR) and is critical for genomic stability. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, we show that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a classic nonhomologous end joining factor, antagonizes double strand break resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1). Autophosphorylation of DNA-PKcs promotes DNA-PKcs dissociation and consequently Exo1 binding. Ataxia telangiectasia-mutated kinase activity can compensate for DNA-PKcs autophosphorylation and promote resection under conditions where DNA-PKcs catalytic activity is inhibited. The Mre11-Rad50-Nbs1 (MRN) complex further stimulates resection in the presence of Ku and DNA-PKcs by recruiting Exo1 and enhancing DNA-PKcs autophosphorylation, and it also inhibits DNA ligase IV/XRCC4-mediated end rejoining. This work suggests that, in addition to its key role in nonhomologous end joining, DNA-PKcs also acts in concert with MRN and ataxia telangiectasia-mutated to regulate resection and thus DNA repair pathway choice.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA por Junção de Extremidades/fisiologia , Enzimas Reparadoras do DNA/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Hidrolases Anidrido Ácido , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Ligase Dependente de ATP , DNA Ligases/genética , DNA Ligases/metabolismo , Enzimas Reparadoras do DNA/genética , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Células HEK293 , Humanos , Autoantígeno Ku , Proteína Homóloga a MRE11 , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , FosforilaçãoRESUMO
BACKGROUND: Conformational selection plays a key role in the polymerase cycle. RESULTS: Klentaq1 exists in conformational equilibrium between three states (open, closed, and "nucleotide-binding") whose level of occupancy is determined by the bound substrate. CONCLUSION: The "nucleotide-binding" state plays a pivotal role in the reaction pathway. SIGNIFICANCE: Direct evidence is provided for the role of a conformationally distinct "nucleotide-binding" state during dNTP incorporation. DNA polymerases are responsible for the accurate replication of DNA. Kinetic, single-molecule, and x-ray studies show that multiple conformational states are important for DNA polymerase fidelity. Using high precision FRET measurements, we show that Klentaq1 (the Klenow fragment of Thermus aquaticus DNA polymerase 1) is in equilibrium between three structurally distinct states. In the absence of nucleotide, the enzyme is mostly open, whereas in the presence of DNA and a correctly base-pairing dNTP, it re-equilibrates to a closed state. In the presence of a dNTP alone, with DNA and an incorrect dNTP, or in elevated MgCl2 concentrations, an intermediate state termed the "nucleotide-binding" state predominates. Photon distribution and hidden Markov modeling revealed fast dynamic and slow conformational processes occurring between all three states in a complex energy landscape suggesting a mechanism in which dNTP delivery is mediated by the nucleotide-binding state. After nucleotide binding, correct dNTPs are transported to the closed state, whereas incorrect dNTPs are delivered to the open state.
Assuntos
Proteínas de Bactérias/química , DNA Polimerase I/química , Thermus/enzimologia , Domínio Catalítico , Nucleotídeos de Desoxiadenina/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Hidrazinas/química , Cinética , Modelos Moleculares , Ligação Proteica , Coloração e Rotulagem , Especificidade por Substrato , Nucleotídeos de Timina/químicaRESUMO
The RecA protein of Deinococcus radiodurans (DrRecA) has a central role in genome reconstitution after exposure to extreme levels of ionizing radiation. When bound to DNA, filaments of DrRecA protein exhibit active and inactive states that are readily interconverted in response to several sets of stimuli and conditions. At 30 °C, the optimal growth temperature, and at physiological pH 7.5, DrRecA protein binds to double-stranded DNA (dsDNA) and forms extended helical filaments in the presence of ATP. However, the ATP is not hydrolyzed. ATP hydrolysis of the DrRecA-dsDNA filament is activated by addition of single-stranded DNA, with or without the single-stranded DNA-binding protein. The ATPase function of DrRecA nucleoprotein filaments thus exists in an inactive default state under some conditions. ATPase activity is thus not a reliable indicator of DNA binding for all bacterial RecA proteins. Activation is effected by situations in which the DNA substrates needed to initiate recombinational DNA repair are present. The inactive state can also be activated by decreasing the pH (protonation of multiple ionizable groups is required) or by addition of volume exclusion agents. Single-stranded DNA-binding protein plays a much more central role in DNA pairing and strand exchange catalyzed by DrRecA than is the case for the cognate proteins in Escherichia coli. The data suggest a mechanism to enhance the efficiency of recombinational DNA repair in the context of severe genomic degradation in D. radiodurans.
Assuntos
Proteínas de Bactérias/metabolismo , Deinococcus/metabolismo , Nucleoproteínas/metabolismo , Recombinases Rec A/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Modelos Biológicos , Ligação Proteica , Estrutura Secundária de Proteína , Recombinases Rec A/antagonistas & inibidores , Temperatura , Fatores de TempoRESUMO
We formulated a master equation-based mathematical model to analyze random scanning and catalysis for enzymes that act on single-stranded DNA (ssDNA) substrates. Catalytic efficiencies and intrinsic scanning distances are deduced from the distribution of positions and gap lengths between a series of catalytic events occurring over time, which are detected as point mutations in a lacZα-based reporter sequence containing enzyme target motifs. Mathematical analysis of the model shows how scanning motions become separable from the catalysis when the proper statistical properties of the mutation pattern are used to interpret the readouts. Two-point correlations between all catalytic events determine intrinsic scanning distances, whereas gap statistics between mutations determine their catalytic efficiencies. Applying this model to activation-induced deoxycytidine deaminase (AID), which catalyzes CâU deaminations processively on ssDNA, we have established that deaminations of AGC hot motifs occur at a low rate, â¼0.03 s(-1), and low efficiency, â¼3%. AID performs random bidirectional movements for an average distance of 6.2 motifs, at a rate of about 15 nucleotides per second, and "dwells" at a motif site for 2.7 s while bound >4 min to the same DNA molecule. These results provide new and important insights on how AID may be optimized for generating mutational diversity in Ig genes, and we discuss how the properties of AID acting freely on a "naked" ssDNA relate to the constrained action of AID during transcription-dependent somatic hypermutation and class-switch recombination.
Assuntos
Algoritmos , Citidina Desaminase/metabolismo , DNA de Cadeia Simples/metabolismo , Modelos Biológicos , Biocatálise , DNA de Cadeia Simples/genética , Cinética , Mutação , Especificidade por Substrato , Transcrição GênicaRESUMO
The Tim-Tipin complex plays an important role in the S phase checkpoint and replication fork stability in metazoans, but the molecular mechanism underlying its biological function is poorly understood. Here, we present evidence that the recombinant human Tim-Tipin complex (and Tim alone) markedly enhances the synthetic activity of DNA polymerase ε. In contrast, no significant effect on the synthetic ability of human DNA polymerase α and δ by Tim-Tipin was observed. Surface plasmon resonance measurements and co-immunoprecipitation experiments revealed that recombinant DNA polymerase ε directly interacts with either Tim or Tipin. In addition, the results of DNA band shift assays suggest that the Tim-Tipin complex (or Tim alone) is able to associate with DNA polymerase ε bound to a 40-/80-mer DNA ligand. Our results are discussed in view of the molecular dynamics at the human DNA replication fork.
Assuntos
Proteínas de Transporte , DNA Polimerase II , DNA , Complexos Multiproteicos , Proteínas Nucleares , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , DNA/biossíntese , DNA/química , DNA/genética , DNA Polimerase II/química , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Proteínas de Ligação a DNA , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ressonância de Plasmônio de Superfície/métodosRESUMO
In this study, we employed a circular replication substrate with a low priming site frequency (1 site/1.1 kb) to quantitatively examine the size distribution and formation pattern of Okazaki fragments. Replication reactions by the T4 replisome on this substrate yielded a patterned series of Okazaki fragments whose size distribution shifted through collision and signaling mechanisms as the gp44/62 clamp loader levels changed but was insensitive to changes in the gp43 polymerase concentration, as expected for a processive, recycled lagging-strand polymerase. In addition, we showed that only one gp45 clamp is continuously associated with the replisome and that no additional clamps accumulate on the DNA, providing further evidence that the clamp departs, whereas the polymerase is recycled upon completion of an Okazaki fragment synthesis cycle. We found no support for the participation of a third polymerase in Okazaki fragment synthesis.
Assuntos
Bacteriófago T4/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , DNA/química , Holoenzimas/metabolismo , Complexos Multienzimáticos/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Modelos Biológicos , Transdução de Sinais , Proteínas Virais/metabolismoRESUMO
Four different type IV secretion systems are variously represented in the genomes of different Helicobacter pylori strains. Two of these, encoded by tfs3 and tfs4 gene clusters are contained within self-transmissible genomic islands. Although chromosomal excision of tfs4 circular intermediates is reported to be dependent upon the function of a tfs4-encoded XerD tyrosine-like recombinase, other factors required for transfer to a recipient cell have not been demonstrated. Here, we characterize the functional activity of a putative tfs4-encoded VirD2-like relaxase protein. Tfs4 VirD2 was purified as a fusion to maltose-binding protein and demonstrated to bind and nick both supercoiled duplex DNA and oligonucleotides in vitro in a manner dependent upon the presence of Mg(2+) but independently of any auxiliary proteins. Unusually, concentration-dependent nicking of duplex DNA appeared to require only transient protein-DNA interaction. Although phylogenetically distinct from established relaxase families, site-specific cleavage of oligonucleotides by Tfs4 VirD2 required the nick region sequence 5'-ATCCTG-3' common to transfer origins (oriT) recognized by MOBP conjugative relaxases. Cleavage resulted in covalent attachment of MBP-VirD2 to the 5'-cleaved end, consistent with conventional relaxase activity. Identification of an oriT-like sequence upstream of tfs4 virD2 and demonstration of VirD2 protein-protein interaction with a putative VirC1 relaxosome component indicate that transfer initiation of the tfs4 genomic island is analogous to mechanisms underlying mobilization of other integrated mobile elements, such as integrating conjugative elements, requiring site-specific targeting of relaxase activity to a cognate oriT sequence.