Function and Structure Relationships in Dna Polymerases

Joyce, Catherine M.; Steitz, Thomas A.

doi:10.1146/annurev.bi.63.070194.004021

Cited by 598 publications

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“…The palm, fingers and thumb domains, which are clearly recognizable in RB69 pol, are common features shared by many DNA polymerases. The catalytic center for the phosphoryl transfer reaction lies in the palm, the most highly conserved domain among all the polymerases (30). Amino acid residues that comprise the nascent base-pair binding pocket of RB69 pol in the "closed" conformation have been identified and are shown in Figure 1 (29).…”

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confidence: 99%

The L561A Substitution in the Nascent Base-Pair Binding Pocket of RB69 DNA Polymerase Reduces Base Discrimination

et al. 2006

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Several variants of RB69 DNA polymerase (RB69 pol) with single-site replacements in the nascent base-pair binding pocket are less discriminating with respect to non-complementary dNMP incorporation than the wild-type enzyme. To quantify the loss in base selectivity, we determined the transient-state kinetic parameters for incorporation of correct and all combinations of incorrect dNMPs by the exonuclease deficient form of one of these RB69 pol variants, L561A, using rapid chemical quench assays. The L561A variant did not significantly alter the k pol and K D values for incorporation of correct dNMPs, but it showed increased incorporation efficiency (k pol / K D ) for mispaired bases relative to the wild type enzyme. The incorporation efficiency for mispaired bases by the L561A variant ranged from 1.5 × 10 −5 µM −1 s −1 for dCMP opposite templating C to 2 × 10 −3 µM −1 s −1 for dAMP opposite templating C. These k pol /K D values are 3-60 fold greater than those observed with the wild type enzyme. The effect of the L561A replacement on the mutation frequency in vivo was determined by infecting E. coli, harboring a plasmid encoding the L561A variant of RB69 pol, with T4 phage bearing a mutant rII locus and the rates of reversions to rII + were scored. The exonuclease-proficient RB69 pol L561A displayed a weak mutator phenotype. In contrast, no progeny phage were produced after infection of E. coli, expressing an exonuclease-deficient RB69 pol L561A, with either mutant or wild type T4 phage. This dominant-lethal phenotype was attributed to error catastrophe caused by the high rate of mutation expected from combining the pol L561A and exo − mutator activities. Keywords base selectivity; transient-state kinetic parameters; mutation frequency; modeling of mismatches DNA polymerase is the central component of replicases that are responsible for faithfully copying DNA. Despite the fact that all four dNTPs are potential substrates, replicative DNA polymerases are able to limit the incorporation of mismatched bases to about one in a million [for reviews see (1-8)]. Although this error frequency is acceptable for phage replication, it is still far too high to maintain genetic integrity during cell proliferation in † Supported by National Institutes of Health, Public Health Service grants GM63276 (to W.K.) and TW006626 (to A.B.). J.W.D. was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences. * To whom correspondence and reprint requests should be addressed. telephone, (203) 785-4599; fax, (203) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript more complex organisms. To increase fidelity, cells have a number of mechanisms for correcting errors, including the ability of the replicative DNA polymerase itself, or an associated subunit, to excise misincorporated bases. In addition, there are specialized DNA polymerases that confront DNA damage inflicted by radiation, oxidation, alkylating agents, etc. which block the progress of replic...

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The L561A Substitution in the Nascent Base-Pair Binding Pocket of RB69 DNA Polymerase Reduces Base Discrimination

et al. 2006

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“…Considering the above examples, it may be general that the complex of the leadzyme and the substrate containing the internal loop, in which the rare earth ion and Pb 2+ could bind to (8) the different purine residues, has two binding sites of the metal ions. Although the roles of the rare earth ions remain unclear based on the present results only, either Pb 2+ or the rare earth ion may activate the attacking water or sugar hydroxyl group, while the other ion stabilizes the oxygen leaving ~[ group such as in the mechanism of the phosphoryl transfer ~ reaction catalyzed by the Y,5'-exonuclease of DNA polymerase I [20]. In fact, NMR has demonstrated the possibility that 5 purine residues in the internal loop near the active site do not form stable non-Watson-Crick base pairs under low salt conditions like 0.1 M NaC1 and hence heterocyclic nitrogens of the bases to which metal ions can bind directly may be free [21,22].…”

Section: Discussionmentioning

confidence: 76%

Site‐specific cleavage reaction catalyzed by leadzyme is enhanced by combined effect of lead and rare earth ions

Sugimoto

Ohmichi

1996

FEBS Letters

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Lead-dependent ribozyme (leadzyme) is a ribozyme working with Pb z+. In this paper, we have investigated the combined effect of metal ions, especially rare earth ions, on the cleavage reaction by the leadzyme. As a result, it was observed that although only a rare earth ion or another divalent ion except Pb z+ did not play a role as the catalyst, the addition of a rare earth ion in the presence of Pb z+ increased significantly the yield of the cleavage reaction. The result suggests that the complex between the leadzyme and the substrate should have two classes of metal ion binding sites.

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“…Dentro de los motivos de transcriptasa reversa, existe una tríada de ácidos aspárticos localizados en los motivos A y C que son indispensables para la actividad pues coordinan los cationes divalentes (Mg ++ ) que se requieren para la catálisis (19,(46)(47)(48). Mutaciones en cualquiera de estos tres ácidos aspárticos de las TERT de levaduras, humano o Tetrahymena, eliminan la actividad de la telomerasa tanto in vitro como in vivo (16,21,29,(48)(49)(50).…”

Section: Discussionunclassified

Identificación de la secuencia del gen de la subunidad catalítica de la telomerasa en Plasmodium falciparum.

Rubiano¹,

Wasserman²

2005

biomedica

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Introducción. La enzima telomerasa participa en la regulación de la longitud de los telómeros al sintetizar nuevas repeticiones teloméricas que compensan las pérdidas en cada ronda de replicación del ADN. Por esta razón, el bloqueo de su actividad se plantea como un posible blanco de acción para detener el crecimiento de células con altas tasas de crecimiento. Tal es el caso de Plasmodium falciparum, parásito causante de la forma más grave de paludismo humano, en el cual se sabe que hay actividad de telomerasa pero no se tiene información sobre la enzima misma. Metodología. Para hacer un acercamiento al estudio de la telomerasa en P. falciparum, se realizó un alineamiento múltiple de las secuencias de la subunidad catalítica de la telomerasa disponibles en bases de datos y se obtuvo una secuencia consenso, la cual se comparó con las secuencias generadas en el proyecto de genoma de P. falciparum. Se encontró una secuencia que podría corresponder a parte del gen de la telomerasa de P. falciparum. Para comprobarlo, se diseñaron iniciadores que se utilizaron en ensayos de amplificación sobre el ADN y el ARN del parásito. Resultados. Se amplificaron fragmentos de ADN correspondientes a motivos conservados en las telomerasas y se detectó la presencia del ARNm mediante trascripción reversa y PCR sobre el ADNc generado. De esta manera, al combinar la utilización de herramientas de bioinformática y su posterior comprobación mediante técnicas de biología molecular, se obtuvo la secuencia del gen de la subunidad catalítica de la telomerasa en P. falciparum y se comprobó su presencia y trascripción en el parásito.Palabras clave: telomerasa, telómero, Plasmodium falciparum, biología computacional, paludismo. Identification of the gene sequence of telomerase catalytic subunit in Plasmodium falciparumIntroduction. The enzyme telomerase regulates telomere length by synthesis of telomeric repeats to compensate for telomeric loss in each DNA replication cycle. Therefore, telomerase is a potential target to block growth of cells with high replication rates. In Plasmodium falciparum, telomerase activity has been documented, but little information on its structure and role. Methods. Herein, alignment of multiple sequences was undertaken comparing telomerase catalytic subunit sequences as found in existing databases. A consensus sequence was compared with the sequences in the P. falciparum genome project and as a result, a candidate sequence for a portion of the telomerase gene was recovered. Primer sets were designed for DNA and RNA amplifications. Results. DNA fragments corresponding to telomerase conserved domains were amplified by using reverse transcription and PCR of cDNA. With a combination of bioinformatics and sequencing methods, the sequence of telomerase catalytic subunit gene (TERT) in P. falciparum was discovered, and its presence and transcription demonstrated.

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Function and Structure Relationships in Dna Polymerases

Cited by 598 publications

References 0 publications

The L561A Substitution in the Nascent Base-Pair Binding Pocket of RB69 DNA Polymerase Reduces Base Discrimination

The L561A Substitution in the Nascent Base-Pair Binding Pocket of RB69 DNA Polymerase Reduces Base Discrimination

Site‐specific cleavage reaction catalyzed by leadzyme is enhanced by combined effect of lead and rare earth ions

Identificación de la secuencia del gen de la subunidad catalítica de la telomerasa en Plasmodium falciparum.

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