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1.
Biochemistry ; 56(21): 2651-2662, 2017 05 30.
Artículo en Inglés | MEDLINE | ID: mdl-28505413

RESUMEN

The herpes helicase-primase (UL5-UL8-UL52) very inefficiently unwinds double-stranded DNA. To better understand the mechanistic consequences of this inefficiency, we investigated protein displacement activity by UL5-UL8-UL52, as well as the impact of coupling DNA synthesis by the herpes polymerase with helicase activity. While the helicase can displace proteins bound to the lagging strand template, bound proteins significantly impede helicase activity. Remarkably, UL5-UL8-UL52, an extremely inefficient helicase, disrupts the exceptionally tight interaction between streptavidin and biotin on the lagging strand template. It also unwinds DNA containing streptavidin bound to the leading strand template, although it does not displace the streptavidin. These data suggest that the helicase may largely or completely wrap around the lagging strand template, with minimal interactions with the leading strand template. We utilized synthetic DNA minicircles to study helicase activity coupled with the herpes polymerase-processivity factor (UL30-UL42). Coupling greatly enhances unwinding of DNA, although bound proteins still inhibit helicase activity. Surprisingly, while UL30-UL42 and two noncognate polymerases (Klenow Fragment and T4 DNA polymerase) all stimulate unwinding of DNA by the helicase, the isolated UL30 polymerase (i.e., no UL42 processivity factor) binds to the replication fork but in a manner that is incompetent in terms of coupled helicase-polymerase activity.


Asunto(s)
ADN Helicasas/metabolismo , ADN Primasa/metabolismo , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/biosíntesis , Exodesoxirribonucleasas/metabolismo , Proteínas Virales/metabolismo
2.
J Org Chem ; 82(20): 10803-10811, 2017 10 20.
Artículo en Inglés | MEDLINE | ID: mdl-28282138

RESUMEN

Small molecule/DNA hybrids (SMDHs) have been considered as nanoscale building blocks for engineering 2D and 3D supramolecular DNA assembly. Herein, we report an efficient on-bead amide-coupling approach to prepare SMDHs with multiple oligodeoxynucleotide (ODN) strands. Our method is high yielding under mild and user-friendly conditions with various organic substrates and homo- or mixed-sequenced ODNs. Metal catalysts and moisture- and air-free conditions are not required. The products can be easily analyzed by LC-MS with accurate mass resolution. We also explored nanometer-sized shape-persistent macrocycles as novel multitopic organic linkers to prepare SMDHs. SMDHs bearing up to six ODNs were successfully prepared through the coupling of arylenethynylene macrocycles with ODNs, which were used to mediate the assembly of gold nanoparticles.


Asunto(s)
Amidas/química , ADN/química , Bibliotecas de Moléculas Pequeñas/química , Estructura Molecular , Oligodesoxirribonucleótidos/química
3.
Biochemistry ; 55(7): 1168-77, 2016 Feb 23.
Artículo en Inglés | MEDLINE | ID: mdl-26836009

RESUMEN

We examined the impact of two clinically approved anti-herpes drugs, acyclovir and Forscarnet (phosphonoformate), on the exonuclease activity of the herpes simplex virus-1 DNA polymerase, UL30. Acyclovir triphosphate and Foscarnet, along with the closely related phosphonoacetic acid, did not affect exonuclease activity on single-stranded DNA. Furthermore, blocking the polymerase active site due to either binding of Foscarnet or phosphonoacetic acid to the E-DNA complex or polymerization of acyclovir onto the DNA also had a minimal effect on exonuclease activity. The inability of the exonuclease to excise acyclovir from the primer 3'-terminus results from the altered sugar structure directly impeding phosphodiester bond hydrolysis as opposed to inhibiting binding, unwinding of the DNA by the exonuclease, or transfer of the DNA from the polymerase to the exonuclease. Removing the 3'-hydroxyl or the 2'-carbon from the nucleotide at the 3'-terminus of the primer strongly inhibited exonuclease activity, although addition of a 2'-hydroxyl did not affect exonuclease activity. The biological consequences of these results are twofold. First, the ability of acyclovir and Foscarnet to block dNTP polymerization without impacting exonuclease activity raises the possibility that their effects on herpes replication may involve both direct inhibition of dNTP polymerization and exonuclease-mediated destruction of herpes DNA. Second, the ability of the exonuclease to rapidly remove a ribonucleotide at the primer 3'-terminus in combination with the polymerase not efficiently adding dNTPs onto this primer provides a novel mechanism by which the herpes replication machinery can prevent incorporation of ribonucleotides into newly synthesized DNA.


Asunto(s)
Aciclovir/farmacología , Antivirales/farmacología , Exodesoxirribonucleasas/antagonistas & inhibidores , Foscarnet/farmacología , Herpesvirus Humano 1/enzimología , Modelos Moleculares , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Proteínas Virales/antagonistas & inhibidores , Aciclovir/química , Aciclovir/metabolismo , Antivirales/química , Antivirales/metabolismo , Dominio Catalítico , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Foscarnet/química , Foscarnet/metabolismo , Herpesvirus Humano 1/efectos de los fármacos , Hidrólisis/efectos de los fármacos , Cinética , Estructura Molecular , Inhibidores de la Síntesis del Ácido Nucleico/química , Inhibidores de la Síntesis del Ácido Nucleico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribonucleótidos/química , Ribonucleótidos/metabolismo , Especificidad por Sustrato , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismo
4.
Biochemistry ; 54(2): 240-9, 2015 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-25517265

RESUMEN

The herpes polymerase-processivity factor complex consists of the catalytic UL30 subunit containing both polymerase and proofreading exonuclease activities and the UL42 subunit that acts as a processivity factor. Curiously, the highly active exonuclease has minimal impact on the accumulation of mismatches generated by the polymerase activity. We utilized a series of oligonucleotides of defined sequence to define the interactions between the polymerase and exonuclease active sites. Exonuclease activity requires unwinding of two nucleotides of the duplex primer-template. Surprisingly, even though the exonuclease rate is much higher than the rate of DNA dissociation, the exonuclease degrades both single- and double-stranded DNA in a nonprocessive manner. Efficient proofreading of incorrect nucleotides incorporated by the polymerase would seem to require efficient translocation of DNA between the exonuclease and polymerase active sites. However, we found that translocation of DNA from the exonuclease to polymerase active site is remarkably inefficient. Consistent with inefficient translocation, the DNA binding sites for the exonuclease and polymerase active sites appear to be largely independent, such that the two activities appear noncoordinated. Finally, the presence or absence of UL42 did not impact the coordination of the polymerase and exonuclease activities. In addition to providing fundamental insights into how the polymerase and exonuclease function together, these activities provide a rationale for understanding why the exonuclease minimally impacts accumulation of mismatches by the purified polymerase and raise the question of how these two activities function together in vivo.


Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Herpes Simple/virología , Herpesvirus Humano 1/enzimología , Proteínas Virales/metabolismo , Dominio Catalítico , ADN/metabolismo , ADN Polimerasa Dirigida por ADN/química , Exodesoxirribonucleasas/química , Herpesvirus Humano 1/química , Herpesvirus Humano 1/metabolismo , Humanos , Modelos Moleculares , Proteínas Virales/química
5.
Chemistry ; 20(7): 2010-5, 2014 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-24311229

RESUMEN

Tricyclic cytosines (tC and tC(O) frameworks) have emerged as a unique class of fluorescent nucleobase analogues that minimally perturb the structure of B-form DNA and that are not quenched in duplex nucleic acids. Systematic derivatization of these frameworks is a likely approach to improve on and diversify photophysical properties, but has not so far been examined. Synthetic methods were refined to improve on tolerance for electron-donating and electron-withdrawing groups, resulting in a series of eight new, fluorescent cytidine analogues. Photophysical studies show that substitution of the framework results in a pattern of effects largely consistent across tC and tC(O) and provides nucleoside fluorophores that are brighter than either parent. Moreover, a range of solvent sensitivities is observed, offering promise that this family of probes can be extended to new applications that require reporting on the local environment.


Asunto(s)
Citosina/análogos & derivados , Colorantes Fluorescentes/química , Nucleósidos/química , ADN Forma B/análisis , Conformación de Ácido Nucleico , Solventes/química
6.
ACS Appl Bio Mater ; 7(8): 5308-5317, 2024 Aug 19.
Artículo en Inglés | MEDLINE | ID: mdl-38978451

RESUMEN

Modulating molecular structure and function at the nanoscale drives innovation across wide-ranging technologies. Electrical control of the bonding of individual DNA base pairs endows DNA with precise nanoscale structural reconfigurability, benefiting efforts in DNA origami and actuation. Here, alloxazine DNA base surrogates were synthesized and incorporated into DNA duplexes to function as a redox-active switch of hydrogen bonding. Circular dichroism (CD) revealed that 24-mer DNA duplexes containing one or two alloxazines exhibited CD spectra and melting transitions similar to DNA with only canonical bases, indicating that the constructs adopt a B-form conformation. However, duplexes were not formed when four or more alloxazines were incorporated into a 24-mer strand. Thiolated duplexes incorporating alloxazines were self-assembled onto multiplexed gold electrodes and probed electrochemically. Square-wave voltammetry (SWV) revealed a substantial reduction peak centered at -0.272 V vs Ag/AgCl reference. Alternating between alloxazine oxidizing and reducing conditions modulated the SWV peak in a manner consistent with the formation and loss of hydrogen bonding, which disrupts the base pair stacking and redox efficiency of the DNA construct. These alternating signals support the assertion that alloxazine can function as a redox-active switch of hydrogen bonding, useful in controlling DNA and bioinspired assemblies.


Asunto(s)
ADN , Enlace de Hidrógeno , Oxidación-Reducción , ADN/química , Ensayo de Materiales , Flavinas/química , Materiales Biocompatibles/química , Materiales Biocompatibles/síntesis química , Tamaño de la Partícula , Conformación de Ácido Nucleico , Estructura Molecular , Técnicas Electroquímicas
7.
J Am Chem Soc ; 135(4): 1205-8, 2013 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-23316816

RESUMEN

To better understand the energetics of accurate DNA replication, we directly measured ΔG(o) for the incorporation of a nucleotide into elongating dsDNA in solution (ΔG(o)(incorporation)). Direct measurements of the energetic difference between synthesis of correct and incorrect base pairs found it to be much larger than previously believed (average ΔΔG(o)(incorporation) = 5.2 ± 1.34 kcal mol(-1)). Importantly, these direct measurements indicate that ΔΔG(o)(incorporation) alone can account for the energy required for highly accurate DNA replication. Evolutionarily, these results indicate that the earliest polymerases did not have to evolve sophisticated mechanisms to replicate nucleic acids; they may only have had to take advantage of the inherently more favorable ΔG(o) for polymerization of correct nucleotides. These results also provide a basis for understanding how polymerases replicate DNA (or RNA) with high fidelity.


Asunto(s)
ADN/química , Termodinámica , Emparejamiento Base , Replicación del ADN
8.
J Virol ; 85(2): 957-67, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21068232

RESUMEN

The origin-specific replication of the herpes simplex virus 1 genome requires seven proteins: the helicase-primase (UL5-UL8-UL52), the DNA polymerase (UL30-UL42), the single-strand DNA binding protein (ICP8), and the origin-binding protein (UL9). We reconstituted these proteins, excluding UL9, on synthetic minicircular DNA templates and monitored leading and lagging strand DNA synthesis using the strand-specific incorporation of dTMP and dAMP. Critical features of the assays that led to efficient leading and lagging stand synthesis included high helicase-primase concentrations and a lagging strand template whose sequence resembled that of the viral DNA. Depending on the nature of the minicircle template, the replication complex synthesized leading and lagging strand products at molar ratios varying between 1:1 and 3:1. Lagging strand products (∼0.2 to 0.6 kb) were significantly shorter than leading strand products (∼2 to 10 kb), and conditions that stimulated primer synthesis led to shorter lagging strand products. ICP8 was not essential; however, its presence stimulated DNA synthesis and increased the length of both leading and lagging strand products. Curiously, human DNA polymerase α (p70-p180 or p49-p58-p70-p180), which improves the utilization of RNA primers synthesized by herpesvirus primase on linear DNA templates, had no effect on the replication of the minicircles. The lack of stimulation by polymerase α suggests the existence of a macromolecular assembly that enhances the utilization of RNA primers and may functionally couple leading and lagging strand synthesis. Evidence for functional coupling is further provided by our observations that (i) leading and lagging strand synthesis produce equal amounts of DNA, (ii) leading strand synthesis proceeds faster under conditions that disable primer synthesis on the lagging strand, and (iii) conditions that accelerate helicase-catalyzed DNA unwinding stimulate decoupled leading strand synthesis but not coordinated leading and lagging strand synthesis.


Asunto(s)
Replicación del ADN , ADN Circular/metabolismo , Herpesvirus Humano 1/enzimología , Proteínas Virales/metabolismo , Cartilla de ADN/genética , ADN Circular/genética , ADN Viral/genética , ADN Viral/metabolismo , Nucleótidos de Desoxiadenina/metabolismo , Moldes Genéticos , Timidina Monofosfato/metabolismo , Proteínas Virales/aislamiento & purificación
9.
J Virol ; 85(2): 968-78, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21068246

RESUMEN

The heterotrimeric helicase-primase complex of herpes simplex virus type I (HSV-1), consisting of UL5, UL8, and UL52, possesses 5' to 3' helicase, single-stranded DNA (ssDNA)-dependent ATPase, primase, and DNA binding activities. In this study we confirm that the UL5-UL8-UL52 complex has higher affinity for forked DNA than for ssDNA and fails to bind to fully annealed double-stranded DNA substrates. In addition, we show that a single-stranded overhang of greater than 6 nucleotides is required for efficient enzyme loading and unwinding. Electrophoretic mobility shift assays and surface plasmon resonance analysis provide additional quantitative information about how the UL5-UL8-UL52 complex associates with the replication fork. Although it has previously been reported that in the absence of DNA and nucleoside triphosphates the UL5-UL8-UL52 complex exists as a monomer in solution, we now present evidence that in the presence of forked DNA and AMP-PNP, higher-order complexes can form. Electrophoretic mobility shift assays reveal two discrete complexes with different mobilities only when helicase-primase is bound to DNA containing a single-stranded region, and surface plasmon resonance analysis confirms larger amounts of the complex bound to forked substrates than to single-overhang substrates. Furthermore, we show that primase activity exhibits a cooperative dependence on protein concentration while ATPase and helicase activities do not. Taken together, these data suggest that the primase activity of the helicase-primase requires formation of a dimer or higher-order structure while ATPase activity does not. Importantly, this provides a simple mechanism for generating a two-polymerase replisome at the replication fork.


Asunto(s)
ADN Helicasas/metabolismo , ADN Primasa/metabolismo , ADN de Cadena Simple/metabolismo , ADN/metabolismo , Herpesvirus Humano 1/enzimología , Multimerización de Proteína , Proteínas Virales/metabolismo , Animales , Ensayo de Cambio de Movilidad Electroforética , Unión Proteica , Resonancia por Plasmón de Superficie
10.
Biochemistry ; 50(33): 7243-50, 2011 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-21761848

RESUMEN

We utilized a series of pyrimidine analogues modified at O(2), N-3, and N(4)/O(4) to determine if two B family DNA polymerases, human DNA polymerase α and herpes simplex virus I DNA polymerase, choose whether to polymerize pyrimidine dNTPs using the same mechanisms they use for purine dNTPs. Removing O(2) of a pyrimidine dNTP vastly decreased the level of incorporation by these enzymes and also compromised fidelity in the case of C analogues, while removing O(2) from the templating base had more modest effects. Removing the Watson-Crick hydrogen bonding groups of N-3 and N(4)/O(4) greatly impaired polymerization, both of the resulting dNTP analogues and of natural dNTPs opposite these pyrimidine analogues when present in the template strand. Thus, the Watson-Crick hydrogen bonding groups of a pyrimidine clearly play an important role in enhancing correct dNTP polymerization but are not essential for preventing misincorporation. These studies also indicate that DNA polymerases recognize bases extremely asymmetrically, both in terms of whether they are a purine or pyrimidine and whether they are in the template or are the incoming dNTP. The mechanistic implications of these results with regard to how polymerases discriminate between right and wrong dNTPs are discussed.


Asunto(s)
ADN Polimerasa I/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Purinas/metabolismo , Pirimidinas/metabolismo , Proteínas Virales/metabolismo , ADN Polimerasa I/química , Replicación del ADN , ADN Polimerasa Dirigida por ADN/química , Exodesoxirribonucleasas/química , Humanos , Enlace de Hidrógeno , Polimerizacion , Purinas/química , Pirimidinas/química , Proteínas Virales/química
11.
Biochim Biophys Acta ; 1804(5): 1180-9, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-19540940

RESUMEN

DNA primase synthesizes short RNA primers that replicative polymerases further elongate in order to initiate the synthesis of all new DNA strands. Thus, primase owes its existence to the inability of DNA polymerases to initiate DNA synthesis starting with 2 dNTPs. Here, we discuss the evolutionary relationships between the different families of primases (viral, eubacterial, archael, and eukaryotic) and the catalytic mechanisms of these enzymes. This includes how they choose an initiation site, elongate the growing primer, and then only synthesize primers of defined length via an inherent ability to count. Finally, the low fidelity of primases along with the development of primase inhibitors is described.


Asunto(s)
ADN Primasa/genética , Evolución Molecular , Secuencia de Aminoácidos , Animales , Cartilla de ADN/genética , Cartilla de ADN/metabolismo , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido
12.
Anal Biochem ; 416(1): 53-60, 2011 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-21600183

RESUMEN

The cytosine analogs 1,3-diaza-2-oxophenothiazine (tC) and 1,3-diaza-2-oxophenoxazine (tCo) stand out among fluorescent bases due to their unquenched fluorescence emission in double-stranded DNA. Recently, we reported a method for the generation of densely tCo-labeled DNA by polymerase chain reaction (PCR) that relied on the use of the extremely thermostable Deep Vent polymerase. We have now developed a protocol that employs the more commonly used Taq polymerase. Supplementing the PCR with Mn(2+) or Co(2+) ions dramatically increased the amount of tCo triphosphate (dtCoTP) incorporated and, thus, enhanced the brightness of the PCR products. The resulting PCR products could be easily detected in gels based on their intrinsic fluorescence. The Mn(2+) ions modulate the PCR by improving the bypass of template tCo and the overall catalytic efficiency. In contrast to the lower fidelity during tCo bypass, Mn(2+) improved the ability of Taq polymerase to distinguish between dtCoTP and dTTP when copying a template dA. Interestingly, Mn(2+) ions hardly affect the fluorescence emission of tC(o), whereas the coordination of Co(2+) ions with the phosphate groups of DNA and nucleotides statically quenches tC(o) fluorescence with small reciprocal Stern-Vollmer constants of 10-300µM.


Asunto(s)
Biocatálisis , Fluorescencia , Oxazinas/química , Fenotiazinas/química , Reacción en Cadena de la Polimerasa , Polimerasa Taq/metabolismo , Elementos de Transición/química , ADN/análisis , ADN/genética , Humanos , Iones/química , Oxazinas/metabolismo , Fenotiazinas/metabolismo , Sensibilidad y Especificidad , Polimerasa Taq/química
13.
Biochemistry ; 49(47): 10208-15, 2010 Nov 30.
Artículo en Inglés | MEDLINE | ID: mdl-21033726

RESUMEN

The influenza RNA-dependent RNA polymerase (RdRp) both replicates the flu's RNA genome and transcribes its mRNA. Replication occurs de novo; however, initiation of transcription requires a 7-methylguanosine 5'-capped primer that is "snatched" from host mRNA via endonuclease and cap binding functions of the influenza polymerase. A key question is how the virus regulates the relative amounts of transcription and replication. We found that the concentration of a capped cellular mRNA, the concentration of the 5' end of the viral RNA, and the concentration of RdRp all regulate the relative amounts of replication versus transcription. The host mRNA, from which the RdRp snatches its capped primer, acts to upregulate transcription and repress replication. Elevated concentrations of the RdRp itself switch the influenza polymerase toward replication, likely through an oligomerization of the polymerase. The 5' end of the vRNA template both activates replication and inhibits transcription of the vRNA template, thereby indicating that RdRp contains an allosteric binding site for the 5' end of the vRNA template. These data provide insights into the regulation of RdRp throughout the viral life cycle and how it synthesizes the appropriate amounts of viral mRNA and replication products (vRNA and cRNA).


Asunto(s)
Orthomyxoviridae/enzimología , Caperuzas de ARN/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Transcripción Genética , Replicación Viral , Sitio Alostérico/fisiología , ARN Complementario/genética , ARN Mensajero/metabolismo , ARN Viral/metabolismo
14.
Biochemistry ; 49(4): 727-35, 2010 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-20030400

RESUMEN

Human DNA primase synthesizes short RNA primers that DNA polymerase alpha then elongates during the initiation of all new DNA strands. Even though primase misincorporates NTPs at a relatively high frequency, this likely does not impact the final DNA product since the RNA primer is replaced with DNA. We used an extensive series of purine and pyrimidine analogues to provide further insights into the mechanism by which primase chooses whether or not to polymerize a NTP. Primase readily polymerized a size-expanded cytosine analogue, 1,3-diaza-2-oxophenothiazine NTP, across from a templating G but not across from A. The enzyme did not efficiently polymerize NTPs incapable of forming two Watson-Crick hydrogen bonds with the templating base with the exception of UTP opposite purine deoxyribonucleoside. Likewise, primase did not generate base pairs between two nucleotides with altered Watson-Crick hydrogen-bonding patterns. Examining the mechanism of NTP polymerization revealed that human primase can misincorporate NTPs via both template misreading and a primer-template slippage mechanism. Together, these data demonstrate that human primase strongly depends on Watson-Crick hydrogen bonds for efficient nucleotide polymerization, much more so than the mechanistically related herpes primase, and provide insights into the potential roles of primer-template stability and base tautomerization during misincorporation.


Asunto(s)
ADN Primasa/química , ADN Primasa/metabolismo , Nucleótidos/química , Emparejamiento Base , Sitios de Unión , Cartilla de ADN/química , Cartilla de ADN/metabolismo , Humanos , Enlace de Hidrógeno , Cinética , Datos de Secuencia Molecular , Nucleótidos/metabolismo , Especificidad por Sustrato
15.
Anal Chem ; 82(3): 1082-9, 2010 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-20067253

RESUMEN

Fluorescent RNA is an important analytical tool in medical diagnostics, RNA cytochemistry, and RNA aptamer development. We have synthesized the fluorescent ribonucleotide analogue 1,3-diaza-2-oxophenothiazine-ribose-5'-triphosphate (tCTP) and tested it as substrate for T7 RNA polymerase in transcription reactions, a convenient route for generating RNA in vitro. When transcribing a guanine, T7 RNA polymerase incorporates tCTP with 2-fold higher catalytic efficiency than CTP and efficiently polymerizes additional NTPs onto the tC. Remarkably, T7 RNA polymerase does not incorporate tCTP with the same ambivalence opposite guanine and adenine with which DNA polymerases incorporate the analogous dtCTP. While several DNA polymerases discriminated against a d(tC-A) base pair only by factors <10, T7 RNA polymerase discriminates against tC-A base pair formation by factors of 40 and 300 when operating in the elongation and initiation mode, respectively. These catalytic properties make T7 RNA polymerase an ideal tool for synthesizing large fluorescent RNA, as we demonstrated by generating a approximately 800 nucleotide RNA in which every cytosine was replaced with tC.


Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Colorantes Fluorescentes/química , Ribonucleótidos/síntesis química , Proteínas Virales/metabolismo , Emparejamiento Base , Electroforesis en Gel de Poliacrilamida/métodos , Cinética , Ribonucleótidos/química , Ribonucleótidos/metabolismo , Espectrometría de Fluorescencia
16.
Biochemistry ; 48(1): 180-9, 2009 Jan 13.
Artículo en Inglés | MEDLINE | ID: mdl-19072331

RESUMEN

We used a series of dNTP analogues in conjunction with templates containing modified bases to elucidate the role that N(2) of a purine plays during dNTP polymerization by human DNA polymerase alpha. Removing N(2) from dGTP had small effects during correct incorporation opposite C but specifically increased misincorporation opposite A. Adding N(2) to dATP and related analogues had small and variable effects on the efficiency of polymerization opposite T. However, the presence of N(2) greatly enhanced polymerization of these dATP analogues opposite a template C. The ability of N(2) to enhance polymerization opposite C likely results from formation of a hydrogen bond between the purine N(2) and pyrimidine O(2). Even in those cases where formation of a wobble base pair, tautomerization, and/or protonation of the base pair between the incoming dNTP and template base cannot occur (e.g., 2-pyridone.purine (or purine analogue) base pairs), N(2) enhanced formation of the base pair. Importantly, N(2) had similar effects on dNTP polymerization both when added to the incoming purine dNTP and when added to the template base being replicated. The mechanistic implications of these results regarding how pol alpha discriminates between right and wrong dNTPs are discussed.


Asunto(s)
ADN Polimerasa I/química , Desoxirribonucleótidos/química , Purinas/química , 2-Aminopurina/química , Adenina/análogos & derivados , Adenina/química , Emparejamiento Base , Biopolímeros , Citosina/química , Humanos , Enlace de Hidrógeno , Cinética
17.
Biochemistry ; 48(34): 8271-8, 2009 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-19642651

RESUMEN

To better understand how DNA polymerases interact with mutagenic bases, we examined how human DNA polymerase alpha (pol alpha), a B family enzyme, and DNA polymerase from Bacillus stearothermophilus (BF), an A family enzyme, generate adenine:hypoxanthine and adenine:8-oxo-7,8-dihydroguanine (8-oxoG) base pairs. Pol alpha strongly discriminated against polymerizing dATP opposite 8-oxoG, and removing N1, N(6), or N7 further inhibited incorporation, whereas removing N3 from dATP dramatically increased incorporation (32-fold). Eliminating N(6) from 3-deaza-dATP now greatly reduced incorporation, suggesting that incorporation of dATP (analogues) opposite 8-oxoguanine proceeds via a Hoogsteen base pair and that pol alpha uses N3 of a purine dNTP to block this incorporation. Pol alpha also polymerized 8-oxo-dGTP across from a templating A, and removing N(6) from the template adenine inhibited incorporation of 8-oxoG. The effects of N1, N(6), and N7 demonstrated a strong interdependence during formation of adenine:hypoxanthine base pairs by pol alpha, and N3 of dATP again helps prevent polymerization opposite a templating hypoxanthine. BF very efficiently polymerized 8-oxo-dGTP opposite adenine, and N1 and N7 of adenine appear to play important roles. BF incorporates dATP opposite 8-oxoG less efficiently, and modifying N1, N(6), or N7 greatly inhibits incorporation. N(6) and, to a lesser extent, N1 help drive hypoxanthine:adenine base-pair formation by BF. The mechanistic implications of these results showing that different polymerases interact very differently with base lesions are discussed.


Asunto(s)
ADN Polimerasa I/metabolismo , Geobacillus stearothermophilus/enzimología , Guanina/análogos & derivados , Hipoxantina/química , Nucleótidos de Purina/química , Nucleótidos de Purina/metabolismo , Guanina/química , Guanina/metabolismo , Humanos , Hipoxantina/metabolismo , Polímeros/química , Polímeros/metabolismo , Unión Proteica
18.
Biochemistry ; 48(15): 3554-64, 2009 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-19166354

RESUMEN

To accurately replicate its viral genome, the Herpes Simplex Virus 1 (HSV-1) DNA polymerase usually polymerizes the correct natural 2'-deoxy-5'-triphosphate (dNTP) opposite the template base being replicated. We employed a series of purine-dNTP analogues to determine the chemical features of the base necessary for the herpes polymerase to avoid polymerizing incorrect dNTPs. The enzyme uses N-3 to prevent misincorporation of purine dNTPs but does not require N-3 for correct polymerization. A free pair of electrons on N-1 also helps prevent misincorporation opposite A, C, and G and strongly drives polymerization opposite T. N6 contributes a small amount both for preventing misincorporation and for correct polymerization. Within the context of guanine in either the incoming dNTP or the template base being replicated, N2 prevents misincorporation opposite adenine but plays at most a minor role for incorporation opposite C. In contrast, adding N2 to the dNTPs of either adenine, purine, 6-chloropurine, or 1-deazapurine greatly enhances incorporation opposite C, likely via the formation of a hydrogen bond between N2 of the purine and O2 of the pyrimidine. Herpes polymerase is very sensitive to the structure of the base pair at the primer 3'-terminus since eliminating N-1, N-3, or N6 from a purine nucleotide at the primer 3'-terminus interfered with polymerization of the next two dNTPs. The biological and evolutionary implications of these data are discussed.


Asunto(s)
Replicación del ADN , ADN Polimerasa Dirigida por ADN/química , Exodesoxirribonucleasas/química , Herpesvirus Humano 1/enzimología , Nucleótidos de Purina/química , Proteínas Virales/química , Replicación Viral , Emparejamiento Base , ADN Viral/biosíntesis , ADN Viral/química , ADN Polimerasa Dirigida por ADN/fisiología , Exodesoxirribonucleasas/fisiología , Herpesvirus Humano 1/crecimiento & desarrollo , Humanos , Subunidades de Proteína/química , Subunidades de Proteína/fisiología , Proteínas Virales/fisiología
19.
Biochemistry ; 48(43): 10199-207, 2009 Nov 03.
Artículo en Inglés | MEDLINE | ID: mdl-19788334

RESUMEN

The helicase-primase complex from herpes simplex virus-1 contains three subunits, UL5, UL52, and UL8. We generated each of the potential two-subunit complexes, UL5-UL52, UL5-UL8, and UL52-UL8, and used a series of kinetic and photo-cross-linking studies to provide further insights into the roles of each subunit in DNA binding and primer synthesis. UL8 increases the rate of primer synthesis by UL5-UL52 by increasing the rate of primer initiation (two NTPs --> pppNpN), the rate-limiting step in primer synthesis. The UL5-UL8 complex lacked any detectable catalytic activity (DNA-dependent ATPase, primase, or RNA polymerase using a RNA primer-template and NTPs as substrates) but could still bind DNA, indicating that UL52 must provide some key amino acids needed for helicase function. The UL52-UL8 complex lacked detectable DNA-dependent ATPase activity and could not synthesize primers on single-stranded DNA. However, it exhibited robust RNA polymerase activity using a RNA primer-template and NTPs as substrates. Thus, UL52 must contain the entire primase active site needed for phosphodiester bond formation, while UL5 minimally contributes amino acids needed for the initiation of primer synthesis. Photo-cross-linking experiments using single-stranded templates containing 5-iodouracil either before, in, or after the canonical 3'-GPyPy (Py is T or C) initiation site for primer synthesis showed that only UL5 cross-linked to the DNA. This occurred for the UL5-UL52, UL5-UL52-UL8, and UL5-UL8 complexes and whether the reaction mixtures contained NTPs. Photo-cross-linking of a RNA primer-template, the product of primer synthesis, containing 5-iodouracil in the template generated the same apparent cross-linked species.


Asunto(s)
ADN Helicasas/metabolismo , ADN Primasa/metabolismo , ADN/metabolismo , Organofosfatos/metabolismo , Subunidades de Proteína/metabolismo , Proteínas Virales/metabolismo , Animales , Secuencia de Bases , Western Blotting , Dominio Catalítico , Línea Celular , ADN/química , ADN Helicasas/química , ADN Primasa/química , Cartilla de ADN/química , Cartilla de ADN/metabolismo , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Organofosfatos/química , Unión Proteica , Subunidades de Proteína/química , Homología de Secuencia de Ácido Nucleico , Spodoptera , Proteínas Virales/química
20.
Biochemistry ; 48(46): 10866-81, 2009 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-19835416

RESUMEN

Herpes simplex virus-1 primase misincorporates the natural NTPs at frequencies of around one error per 30 NTPs polymerized, making it one of the least accurate polymerases known. We used a series of nucleotide analogues to further test the hypothesis that primase requires Watson-Crick hydrogen bond formation to efficiently polymerize a NTP. Primase could not generate base pairs containing a complete set of hydrogen bonds in an altered arrangement (isoguanine.isocytosine) and did not efficiently polymerize dNTPs completely incapable of forming Watson-Crick hydrogen bonds opposite templating bases incapable of forming Watson-Crick hydrogen bonds. Similarly, primase did not incorporate most NTPs containing hydrophobic bases incapable of Watson-Crick hydrogen bonding opposite natural template bases. However, 2-pyridone NTP and 4-methyl-2-pyridone NTP provided striking exceptions to this rule. The effects of removing single Watson-Crick hydrogen bonding groups from either the NTP or templating bases varied from almost no effect to completely blocking polymerization depending both on the parental base pair (G.C vs A.T/U) and which base pair of the growing primer (second, third, or fourth) was examined. Thus, primase does not absolutely need to form Watson-Crick hydrogen bonds to efficiently polymerize a NTP. Additionally, we found that herpes primase can misincorporate nucleotides both by misreading the template and by a primer-template slippage mechanism. The mechanistic and biological implications of these results are discussed.


Asunto(s)
Biocatálisis , ADN Helicasas/metabolismo , ADN Primasa/metabolismo , Herpesvirus Humano 1/enzimología , Proteínas Virales/metabolismo , Emparejamiento Base , Desoxirribonucleótidos/química , Desoxirribonucleótidos/metabolismo , Enlace de Hidrógeno , Estructura Molecular , Oligodesoxirribonucleótidos , ARN/biosíntesis , Ribonucleótidos/química , Ribonucleótidos/metabolismo , Especificidad por Sustrato
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