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1.
J Reprod Infant Psychol ; 39(4): 382-394, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-32186924

RESUMEN

Objective: The effectiveness of a cognitive behavioural intervention to prevent perinatal depression in low-income Latina immigrant pregnant women and mothers receiving WIC services was evaluated in a mixed methods study using a community based observational design.Background: The Mothers and Babies Course is a preventive intervention for perinatal depression that is based on cognitive behavioural theory (CBT). CBT is an evidence-based treatment and preventive intervention for perinatal depression.Method: Phase 1 includes 86 Latinas, predominantly Central American immigrant women at high risk for depression, who self-selected into the Mothers and Babies Course, a six-week Spanish CBT group intervention aimed at teaching women mood regulation skills to prevent the onset of depression. Participants, who were recruited from the Women, Infants, and Children services, completed measures of depression and psychopathology at pre-, 6 weeks, and 3 months post-intervention. Phase 2 includes qualitative interviews with a randomly selected subsample (n = 26) from Phase 1 to understand the mechanisms and impact of participants' experiences with the intervention and study.Results: Results indicated no significant differences in depressive symptoms among participants with varied attendance levels (0 class; 1-3 classes = non-completers; 4-6 classes = completers). None of the participants met diagnostic criteria for major depressive disorder at the final data collection period. Despite the varied attendance, both quantitative and qualitative results indicated that completers and non-completers reported similar experiences in the intervention and benefiting from study participation.Conclusion: Conducting mixed methods research highlights the complexity of understanding who can benefit from preventive interventions.


Asunto(s)
Trastorno Depresivo Mayor , Emigrantes e Inmigrantes , Niño , Depresión/prevención & control , Trastorno Depresivo Mayor/prevención & control , Femenino , Hispánicos o Latinos , Humanos , Lactante , Madres , Embarazo
2.
J Biol Chem ; 291(12): 6456-70, 2016 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-26797125

RESUMEN

Replicative DNA polymerases (DNAPs) require divalent metal cations for phosphodiester bond formation in the polymerase site and for hydrolytic editing in the exonuclease site. Me(2+) ions are intimate architectural components of each active site, where they are coordinated by a conserved set of amino acids and functional groups of the reaction substrates. Therefore Me(2+) ions can influence the noncovalent transitions that occur during each nucleotide addition cycle. Using a nanopore, transitions in individual Φ29 DNAP complexes are resolved with single-nucleotide spatial precision and sub-millisecond temporal resolution. We studied Mg(2+) and Mn(2+), which support catalysis, and Ca(2+), which supports deoxynucleoside triphosphate (dNTP) binding but not catalysis. We examined their effects on translocation, dNTP binding, and primer strand transfer between the polymerase and exonuclease sites. All three metals cause a concentration-dependent shift in the translocation equilibrium, predominantly by decreasing the forward translocation rate. Me(2+) also promotes an increase in the backward translocation rate that is dependent upon the primer terminal 3'-OH group. Me(2+) modulates the translocation rates but not their response to force, suggesting that Me(2+) does not affect the distance to the transition state of translocation. Absent Me(2+), the primer strand transfer pathway between the polymerase and exonuclease sites displays additional kinetic states not observed at >1 mm Me(2+). Complementary dNTP binding is affected by Me(2+) identity, with Ca(2+) affording the highest affinity, followed by Mn(2+), and then Mg(2+). Both Ca(2+) and Mn(2+) substantially decrease the dNTP dissociation rate relative to Mg(2+), while Ca(2+) also increases the dNTP association rate.


Asunto(s)
Cloruro de Calcio/química , Cloruros/química , ADN Polimerasa Dirigida por ADN/química , Cloruro de Magnesio/química , Compuestos de Manganeso/química , Proteínas Virales/química , Sustitución de Aminoácidos , Bacteriófagos/enzimología , Biocatálisis , Replicación del ADN , Desoxicitidina Monofosfato/química , Nucleótidos de Desoxiguanina/química , Cinética , Polimerizacion , Unión Proteica
3.
Biochemistry ; 53(51): 8061-76, 2014 Dec 30.
Artículo en Inglés | MEDLINE | ID: mdl-25478721

RESUMEN

Ribonucleoside triphosphates (rNTPs) are frequently incorporated during DNA synthesis by replicative DNA polymerases (DNAPs), and once incorporated are not efficiently edited by the DNAP exonucleolytic function. We examined the kinetic mechanisms that govern selection of complementary deoxyribonucleoside triphosphates (dNTPs) over complementary rNTPs and that govern the probability of a complementary ribonucleotide at the primer terminus escaping exonucleolytic editing and becoming stably incorporated. We studied the quantitative responses of individual Φ29 DNAP complexes to ribonucleotides using a kinetic framework, based on our prior work, in which transfer of the primer strand from the polymerase to exonuclease site occurs prior to translocation, and translocation precedes dNTP binding. We determined transition rates between the pre-translocation and post-translocation states, between the polymerase and exonuclease sites, and for dNTP or rNTP binding, with single-nucleotide spatial precision and submillisecond temporal resolution, from ionic current time traces recorded when individual DNAP complexes are held atop a nanopore in an electric field. The predominant response to the presence of a ribonucleotide in Φ29 DNAP complexes before and after covalent incorporation is significant destabilization, relative to the presence of a deoxyribonucleotide. This destabilization is manifested in the post-translocation state prior to incorporation as a substantially higher rNTP dissociation rate and manifested in the pre-translocation state after incorporation as rate increases for both primer strand transfer to the exonuclease site and the forward translocation, with the probability of editing not directly increased. In the post-translocation state, the primer terminal 2'-OH group also destabilizes dNTP binding.


Asunto(s)
ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Fagos de Bacillus/enzimología , Fagos de Bacillus/genética , Cartilla de ADN/química , Cartilla de ADN/metabolismo , Replicación del ADN , ADN Polimerasa Dirigida por ADN/genética , Desoxirribonucleótidos/química , Desoxirribonucleótidos/metabolismo , Cinética , Modelos Biológicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Nanoporos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Virales/genética
4.
J Adolesc ; 37(8): 1227-35, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25238209

RESUMEN

Poor, adolescent, racial/ethnic minority women are at great risk for developing perinatal depression. However, little research has been conducted evaluating interventions for this population. We conducted a systematic review of preventive and treatment interventions for perinatal depression tested with adolescents, with a focus on low income, minority populations. Nine research-based articles (including one that reported on two studies) were reviewed systematically, and quality ratings were assigned based on a validated measure assessing randomization, double-blinding, and reporting of participant withdrawals. Two treatment studies were identified, both of which were successful in reducing depression. Eight prevention studies were located, of which four were more efficacious than control conditions in preventing depression. Studies sampled mostly minority, low socioeconomic status adolescents. No consistent characteristics across efficacious interventions could be identified. This review underscores the need for researchers to further investigate and build an evidence base.


Asunto(s)
Trastorno Depresivo/terapia , Complicaciones del Embarazo/psicología , Embarazo en Adolescencia/psicología , Adolescente , Trastorno Depresivo/complicaciones , Trastorno Depresivo/prevención & control , Femenino , Humanos , Grupos Minoritarios/psicología , Pobreza/psicología , Embarazo , Complicaciones del Embarazo/prevención & control , Complicaciones del Embarazo/terapia
5.
J Am Chem Soc ; 136(19): 7117-31, 2014 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-24761828

RESUMEN

Exonucleolytic editing of incorrectly incorporated nucleotides by replicative DNA polymerases (DNAPs) plays an essential role in the fidelity of DNA replication. Editing requires that the primer strand of the DNA substrate be transferred between the DNAP polymerase and exonuclease sites, separated by a distance that is typically on the order of ~30 Å. Dynamic transitions between functional states can be quantified with single-nucleotide spatial precision and submillisecond temporal resolution from ionic current time traces recorded when individual DNAP complexes are held atop a nanoscale pore in an electric field. In this study, we have exploited this capability to determine the kinetic relationship between the translocation step and primer strand transfer between the polymerase and exonuclease sites in complexes formed between the replicative DNAP from bacteriophage Φ29 and DNA. We demonstrate that the pathway for primer strand transfer from the polymerase to exonuclease site initiates prior to the translocation step, while complexes are in the pre-translocation state. We developed a mathematical method to determine simultaneously the forward and reverse translocation rates and the rates of primer strand transfer in both directions between the polymerase and the exonuclease sites, and we have applied it to determine these rates for Φ29 DNAP complexes formed with a DNA substrate bearing a fully complementary primer-template duplex. This work provides a framework that will be extended to determine the kinetic mechanisms by which incorporation of noncomplementary nucleotides promotes primer strand transfer from the polymerase site to the exonuclease site.


Asunto(s)
Fagos de Bacillus/enzimología , ADN Viral/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Fagos de Bacillus/química , Fagos de Bacillus/genética , Fagos de Bacillus/metabolismo , Secuencia de Bases , Dominio Catalítico , Replicación del ADN , ADN Viral/química , ADN Viral/genética , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , Cinética , Mutación Puntual , Termodinámica
6.
J Biol Chem ; 289(10): 6350-6361, 2014 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-24464581

RESUMEN

The Φ29 DNA polymerase (DNAP) is a processive B-family replicative DNAP. Fluctuations between the pre-translocation and post-translocation states can be quantified from ionic current traces, when individual Φ29 DNAP-DNA complexes are held atop a nanopore in an electric field. Based upon crystal structures of the Φ29 DNAP-DNA binary complex and the Φ29 DNAP-DNA-dNTP ternary complex, residues Tyr-226 and Tyr-390 in the polymerase active site were implicated in the structural basis of translocation. Here, we have examined the dynamics of translocation and substrate binding in complexes formed with the Y226F and Y390F mutants. The Y226F mutation diminished the forward and reverse rates of translocation, increased the affinity for dNTP in the post-translocation state by decreasing the dNTP dissociation rate, and increased the affinity for pyrophosphate in the pre-translocation state. The Y390F mutation significantly decreased the affinity for dNTP in the post-translocation state by decreasing the association rate ∼2-fold and increasing the dissociation rate ∼10-fold, implicating this as a mechanism by which this mutation impedes DNA synthesis. The Y390F dissociation rate increase is suppressed when complexes are examined in the presence of Mn(2+) rather than Mg(2+). The same effects of the Y226F or Y390F mutations were observed in the background of the D12A/D66A mutations, located in the exonuclease active site, ∼30 Å from the polymerase active site. Although translocation rates were unaffected in the D12A/D66A mutant, these exonuclease site mutations caused a decrease in the dNTP dissociation rate, suggesting that they perturb Φ29 DNAP interdomain architecture.


Asunto(s)
Fagos de Bacillus/enzimología , Dominio Catalítico , ADN Viral/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Fagos de Bacillus/genética , Replicación del ADN/genética , ADN Viral/genética , ADN Polimerasa Dirigida por ADN/genética , Mutación , Unión Proteica , Transporte de Proteínas , Especificidad por Sustrato
7.
J Am Chem Soc ; 135(24): 9149-55, 2013 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-23705688

RESUMEN

Complexes formed between phi29 DNA polymerase (DNAP) and DNA fluctuate discretely between the pre-translocation and post-translocation states on the millisecond time scale. The translocation fluctuations can be observed in ionic current traces when individual complexes are captured atop the α-hemolysin nanopore in an electric field. The presence of complementary 2'-deoxynucleoside triphosphate (dNTP) shifts the equilibrium across the translocation step toward the post-translocation state. Here we have determined quantitatively the kinetic relationship between the phi29 DNAP translocation step and dNTP binding. We demonstrate that dNTP binds to phi29 DNAP-DNA complexes only after the transition from the pre-translocation state to the post-translocation state; dNTP binding rectifies the translocation but it does not directly drive the translocation. Based on the measured time traces of current amplitude, we developed a method for determining the forward and reverse translocation rates and the dNTP association and dissociation rates, individually at each dNTP concentration and each voltage. The translocation rates, and their response to force, match those determined for phi29 DNAP-DNA binary complexes and are unaffected by dNTP. The dNTP association and dissociation rates do not vary as a function of voltage, indicating that force does not distort the polymerase active site and that dNTP binding does not directly involve a displacement in the translocation direction. This combined experimental and theoretical approach and the results obtained provide a framework for separately evaluating the effects of biological variables on the translocation transitions and their effects on dNTP binding.


Asunto(s)
Fagos de Bacillus/enzimología , ADN Polimerasa Dirigida por ADN/metabolismo , Nucleótidos/metabolismo , Fagos de Bacillus/metabolismo , Secuencia de Bases , ADN/metabolismo , Cinética
8.
J Am Chem Soc ; 134(45): 18816-23, 2012 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-23101437

RESUMEN

Complexes formed between the bacteriophage phi29 DNA polymerase (DNAP) and DNA fluctuate between the pre-translocation and post-translocation states on the millisecond time scale. These fluctuations can be directly observed with single-nucleotide precision in real-time ionic current traces when individual complexes are captured atop the α-hemolysin nanopore in an applied electric field. We recently quantified the equilibrium across the translocation step as a function of applied force (voltage), active-site proximal DNA sequences, and the binding of complementary dNTP. To gain insight into the mechanism of this step in the DNAP catalytic cycle, in this study, we have examined the stochastic dynamics of the translocation step. The survival probability of complexes in each of the two states decayed at a single exponential rate, indicating that the observed fluctuations are between two discrete states. We used a robust mathematical formulation based on the autocorrelation function to extract the forward and reverse rates of the transitions between the pre-translocation state and the post-translocation state from ionic current traces of captured phi29 DNAP-DNA binary complexes. We evaluated each transition rate as a function of applied voltage to examine the energy landscape of the phi29 DNAP translocation step. The analysis reveals that active-site proximal DNA sequences influence the depth of the pre-translocation and post-translocation state energy wells and affect the location of the transition state along the direction of the translocation.


Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , ADN/metabolismo , Termodinámica , Biocatálisis , ADN/química , ADN Polimerasa Dirigida por ADN/química
9.
J Biol Chem ; 287(16): 13407-21, 2012 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-22378784

RESUMEN

Complexes of phi29 DNA polymerase and DNA fluctuate on the millisecond time scale between two ionic current amplitude states when captured atop the α-hemolysin nanopore in an applied field. The lower amplitude state is stabilized by complementary dNTP and thus corresponds to complexes in the post-translocation state. We have demonstrated that in the upper amplitude state, the DNA is displaced by a distance of one nucleotide from the post-translocation state. We propose that the upper amplitude state corresponds to complexes in the pre-translocation state. Force exerted on the template strand biases the complexes toward the pre-translocation state. Based on the results of voltage and dNTP titrations, we concluded through mathematical modeling that complementary dNTP binds only to the post-translocation state, and we estimated the binding affinity. The equilibrium between the two states is influenced by active site-proximal DNA sequences. Consistent with the assignment of the upper amplitude state as the pre-translocation state, a DNA substrate that favors the pre-translocation state in complexes on the nanopore is a superior substrate in bulk phase for pyrophosphorolysis. There is also a correlation between DNA sequences that bias complexes toward the pre-translocation state and the rate of exonucleolysis in bulk phase, suggesting that during DNA synthesis the pathway for transfer of the primer strand from the polymerase to exonuclease active site initiates in the pre-translocation state.


Asunto(s)
Fagos de Bacillus/enzimología , Fagos de Bacillus/genética , Replicación del ADN/fisiología , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Nanoporos , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Dominio Catalítico/fisiología , ADN Viral/metabolismo , ADN Polimerasa Dirigida por ADN/síntesis química , Difosfatos/metabolismo , Activación Enzimática/fisiología , Exonucleasas/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Secuencias Invertidas Repetidas/genética , Proteínas Motoras Moleculares/fisiología , Conformación de Ácido Nucleico
10.
Nat Biotechnol ; 30(4): 344-8, 2012 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-22334048

RESUMEN

An emerging DNA sequencing technique uses protein or solid-state pores to analyze individual strands as they are driven in single-file order past a nanoscale sensor. However, uncontrolled electrophoresis of DNA through these nanopores is too fast for accurate base reads. Here, we describe forward and reverse ratcheting of DNA templates through the α-hemolysin nanopore controlled by phi29 DNA polymerase without the need for active voltage control. DNA strands were ratcheted through the pore at median rates of 2.5-40 nucleotides per second and were examined at one nucleotide spatial precision in real time. Up to 500 molecules were processed at ∼130 molecules per hour through one pore. The probability of a registry error (an insertion or deletion) at individual positions during one pass along the template strand ranged from 10% to 24.5% without optimization. This strategy facilitates multiple reads of individual strands and is transferable to other nanopore devices for implementation of DNA sequence analysis.


Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Nanoporos , Replicación del ADN/genética , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , Proteínas Hemolisinas/química , Nucleótidos/química , Nucleótidos/genética
11.
J Biol Chem ; 286(16): 14480-92, 2011 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-21362617

RESUMEN

During each catalytic cycle, DNA polymerases select deoxyribonucleoside triphosphate (dNTP) substrates complementary to a templating base with high fidelity from a pool that includes noncomplementary dNTPs and both complementary and noncomplementary ribonucleoside triphosphates (rNTPs). The Klenow fragment of Escherichia coli DNA polymerase I (KF) achieves this through a series of conformational transitions that precede the chemical step of phosphodiester bond formation. Kinetic evidence from fluorescence and FRET experiments indicates that discrimination of the base and sugar moieties of the incoming nucleotide occurs in distinct, sequential steps during the selection pathway. Here we show that KF-DNA complexes formed with complementary rNTPs or with noncomplementary nucleotides can be distinguished on the basis of their properties when captured in an electric field atop the α-hemolysin nanopore. The average nanopore dwell time of KF-DNA complexes increased as a function of complementary rNTP concentration. The increase was less than that promoted by complementary dNTP, indicating that the rNTP complexes are more stable than KF-DNA binary complexes but less stable than KF-DNA-dNTP ternary complexes. KF-DNA-rNTP complexes could also be distinguished from KF-DNA-dNTP complexes on the basis of ionic current amplitude. In contrast to complementary rNTPs, noncomplementary dNTPs and rNTPs diminished the average nanopore dwell time of KF-DNA complexes in a concentration-dependent manner, suggesting that binding of a noncomplementary nucleotide keeps the KF-DNA complex in a less stable state. These results imply that nucleotide selection proceeds through a series of complexes of increasing stability in which substrates with the correct moiety promote the forward transitions.


Asunto(s)
ADN Polimerasa I/química , Nanoporos , Algoritmos , Biofisica/métodos , ADN/química , ADN Polimerasa I/metabolismo , Electrofisiología , Escherichia coli/enzimología , Transferencia Resonante de Energía de Fluorescencia/métodos , Cinética , Modelos Estadísticos , Nanotecnología/métodos , Nucleótidos/química , Oligonucleótidos/química , Unión Proteica , Especificidad por Sustrato
12.
J Am Chem Soc ; 132(50): 17961-72, 2010 Dec 22.
Artículo en Inglés | MEDLINE | ID: mdl-21121604

RESUMEN

Coupling nucleic acid processing enzymes to nanoscale pores allows controlled movement of individual DNA or RNA strands that is reported as an ionic current/time series. Hundreds of individual enzyme complexes can be examined in single-file order at high bandwidth and spatial resolution. The bacteriophage phi29 DNA polymerase (phi29 DNAP) is an attractive candidate for this technology, due to its remarkable processivity and high affinity for DNA substrates. Here we show that phi29 DNAP-DNA complexes are stable when captured in an electric field across the α-hemolysin nanopore. DNA substrates were activated for replication at the nanopore orifice by exploiting the 3'-5' exonuclease activity of wild-type phi29 DNAP to excise a 3'-H terminal residue, yielding a primer strand 3'-OH. In the presence of deoxynucleoside triphosphates, DNA synthesis was initiated, allowing real-time detection of numerous sequential nucleotide additions that was limited only by DNA template length. Translocation of phi29 DNAP along DNA substrates was observed in real time at Ångstrom-scale precision as the template strand was drawn through the nanopore lumen during replication.


Asunto(s)
ADN Polimerasa Dirigida por ADN/química , Nanoporos , Proteínas Virales/química , Catálisis , Replicación del ADN , Modelos Biológicos , Especificidad por Sustrato
13.
Nat Nanotechnol ; 5(11): 798-806, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20871614

RESUMEN

Nanopores can be used to analyse DNA by monitoring ion currents as individual strands are captured and driven through the pore in single file by an applied voltage. Here, we show that serial replication of individual DNA templates can be achieved by DNA polymerases held at the α-haemolysin nanopore orifice. Replication is blocked in the bulk phase, and is initiated only after the DNA is captured by the nanopore. We used this method, in concert with active voltage control, to observe DNA replication catalysed by bacteriophage T7 DNA polymerase (T7DNAP) and by the Klenow fragment of DNA polymerase I (KF). T7DNAP advanced on a DNA template against an 80-mV load applied across the nanopore, and single nucleotide additions were measured on the millisecond timescale for hundreds of individual DNA molecules in series. Replication by KF was not observed when this enzyme was held on top of the nanopore orifice at an applied potential of 80 mV. Sequential nucleotide additions by KF were observed upon applying controlled voltage reversals.


Asunto(s)
Replicación del ADN , ADN/metabolismo , Electroforesis , Nanoporos , Nanotecnología/métodos , Proteínas Bacterianas , ADN/química , ADN Polimerasa I/química , ADN Polimerasa I/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Campos Electromagnéticos , Proteínas Hemolisinas/química , Modelos Moleculares , Oligonucleótidos/química , Oligonucleótidos/metabolismo
14.
ACS Nano ; 3(6): 1457-66, 2009 Jun 23.
Artículo en Inglés | MEDLINE | ID: mdl-19489560

RESUMEN

DNA polymerases are molecular motors that catalyze template-dependent DNA replication, advancing along template DNA by one nucleotide with each catalytic cycle. Nanopore-based measurements have emerged as a single molecule technique for the study of these enzymes. Using the alpha-hemolysin nanopore, we determined the position of DNA templates bearing inserts of abasic (1',2'-dideoxy) residues, bound to the Klenow fragment of Escherichia coli DNA polymerase I (KF) or to bacteriophage T7 DNA polymerase. Hundreds of individual polymerase complexes were analyzed at 5 A precision within minutes. We generated a map of current amplitudes for DNA-KF-deoxynucleoside triphosphate (dNTP) ternary complexes, using a series of templates bearing blocks of three abasic residues that were displaced by approximately 5 A in the nanopore lumen. Plotted as a function of the distance of the abasic insert from n = 0 in the active site of the enzyme held atop the pore, this map has a single peak. The map is similar when the primer length, the DNA sequences flanking the abasic insert, and the DNA sequences in the vicinity of the KF active site are varied. Primer extension catalyzed by KF using a three abasic template in the presence of a mixture of dNTPs and 2',3'-dideoxynucleoside triphosphates resulted in a ladder of ternary complexes with discrete amplitudes that closely corresponded to this map. An ionic current map measured in the presence of 0.15 M KCl mirrored the map obtained with 0.3 M KCl, permitting experiments with a broader range of mesophilic DNA and RNA processing enzymes. We used the abasic templates to show that capture of complexes with the KF homologue, T7 DNA polymerase, yields an amplitude map nearly indistinguishable from the KF map.


Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , ADN/metabolismo , Nanoestructuras , Moldes Genéticos , Secuencia de Bases , Datos de Secuencia Molecular
15.
ACS Nano ; 3(4): 995-1003, 2009 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-19338283

RESUMEN

DNA polymerases catalyze template-dependent genome replication. The assembly of a high affinity ternary complex between these enzymes, the double strand-single strand junction of their DNA substrate, and the deoxynucleoside triphosphate (dNTP) complementary to the first template base in the polymerase active site is essential to this process. We present a single molecule method for iterative measurements of DNA-polymerase complex assembly with high temporal resolution, using active voltage control of individual DNA substrate molecules tethered noncovalently in an alpha-hemolysin nanopore. DNA binding states of the Klenow fragment of Escherichia coli DNA polymerase I (KF) were diagnosed based upon their ionic current signature, and reacted to with submillisecond precision to execute voltage changes that controlled exposure of the DNA substrate to KF and dNTP. Precise control of exposure times allowed measurements of DNA-KF complex assembly on a time scale that superimposed with the rate of KF binding. Hundreds of measurements were made with a single tethered DNA molecule within seconds, and dozens of molecules can be tethered within a single experiment. This approach allows statistically robust analysis of the assembly of complexes between DNA and RNA processing enzymes and their substrates at the single molecule level.


Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , ADN/metabolismo , Toxinas Bacterianas/química , Secuencia de Bases , ADN/química , ADN/genética , ADN Polimerasa I/química , ADN Polimerasa I/metabolismo , ADN Polimerasa Dirigida por ADN/química , Proteínas Hemolisinas/química , Sustancias Macromoleculares , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Nanoestructuras/química , Nanotecnología , Electricidad Estática
16.
J Am Chem Soc ; 131(10): 3772-8, 2009 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-19275265

RESUMEN

Nanoscale pores are a tool for single molecule analysis of DNA or RNA processing enzymes. Monitoring catalytic activity in real time using this technique requires that these enzymes retain function while held atop a nanopore in an applied electric field. Using an alpha-hemolysin nanopore, we measured the dwell time for complexes of DNA with the Klenow fragment of Escherichia coli DNA polymerase I (KF) as a function of the concentration of deoxynucleoside triphosphate (dNTP) substrate. We analyzed these dwell time measurements in the framework of a two-state model for captured complexes (DNA-KF binary and DNA-KF-dNTP ternary states). Average nanopore dwell time increased without saturating as a function of correct dNTP concentration across 4 orders of magnitude. This arises from two factors that are proportional to dNTP concentration: (1) The fraction of complexes that are in the ternary state when initially captured predominantly affects dwell time at low dNTP concentrations. (2) The rate of binding and rebinding of dNTP to captured complexes affects dwell time at higher dNTP concentrations. Thus there are two regimes that display a linear relationship between average dwell time and dNTP concentration. The transition from one linear regime to the other occurs near the equilibrium dissociation constant (K(d)) for dNTP binding to KF-DNA complexes in solution. We conclude from the combination of titration experiments and modeling that DNA-KF complexes captured atop the nanopore retain iterative, sequence-specific dNTP binding, as required for catalysis and fidelity in DNA synthesis.


Asunto(s)
ADN Polimerasa I/metabolismo , Nucleótidos/metabolismo , Replicación del ADN , Nucleótidos de Desoxiguanina/metabolismo , Escherichia coli/enzimología , Unión Proteica
17.
Artículo en Inglés | MEDLINE | ID: mdl-19164022

RESUMEN

This paper demonstrates feedback voltage control of a single DNA molecule tethered in a biological nanopore. The nanopore device monitors ionic current through a single protein pore inserted in a lipid bilayer. The limiting aperture of the pore is just sufficient (1.5 nm diameter) to accommodate single-stranded DNA. The tethered DNA is double stranded on each end, with a single stranded segment that traverses the pore. Voltage control is used to regulate the motion of the tethered DNA, for repeated capture and subsequent voltage-promoted dissociation of DNA-binding enzymes above the nanopore. In initial experiments using the Klenow fragment of Escherichia coli DNA polymerase I, control of 8 independent tethered DNA molecules yielded 337 dissociation events in a period of 380 seconds. The resulting distribution of DNA-protein dissociation times can be used to model the free energy profile of dissociation. Moreover, the approach is applicable to numerous enzymes that bind or modify DNA or RNA including exonucleases, kinases, and other polymerases.


Asunto(s)
ADN Polimerasa Dirigida por ADN/química , ADN/química , Electroquímica/instrumentación , Micromanipulación/instrumentación , Técnicas de Sonda Molecular/instrumentación , Nanotecnología/instrumentación , Mapeo de Interacción de Proteínas/instrumentación , Electroquímica/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Retroalimentación , Membrana Dobles de Lípidos/química , Micromanipulación/métodos , Nanotecnología/métodos , Porosidad , Unión Proteica , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
18.
Nat Nanotechnol ; 2(11): 718-24, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18654412

RESUMEN

Nanoscale pores have potential to be used as biosensors and are an established tool for analysing the structure and composition of single DNA or RNA molecules. Recently, nanopores have been used to measure the binding of enzymes to their DNA substrates. In this technique, a polynucleotide bound to an enzyme is drawn into the nanopore by an applied voltage. The force exerted on the charged backbone of the polynucleotide by the electric field is used to examine the enzyme-polynucleotide interactions. Here we show that a nanopore sensor can accurately identify DNA templates bound in the catalytic site of individual DNA polymerase molecules. Discrimination among unbound DNA, binary DNA/polymerase complexes, and ternary DNA/polymerase/deoxynucleotide triphosphate complexes was achieved in real time using finite state machine logic. This technique is applicable to numerous enzymes that bind or modify DNA or RNA including exonucleases, kinases and other polymerases.


Asunto(s)
Bioensayo/métodos , ADN Polimerasa Dirigida por ADN/química , ADN/química , Nanoestructuras/química , Nanoestructuras/ultraestructura , Nanotecnología/métodos , Secuencia de Aminoácidos , Sitios de Unión , Sustancias Macromoleculares/química , Datos de Secuencia Molecular , Porosidad , Unión Proteica
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