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Videos de Conceptos Relacionados

Translesion DNA Polymerases02:10

Translesion DNA Polymerases

Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
Proofreading01:31

Proofreading

Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore,  it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
Errors During Replication are Corrected by the DNA Polymerase Enzyme
Proofreading01:43

Proofreading

Synthesis of new DNA molecules starts when DNA polymerase links nucleotides together in a sequence that is complementary to the template DNA strand. DNA polymerase has a higher affinity for the correct base to ensure fidelity in DNA replication. The DNA polymerase furthermore proofreads during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.Errors during Replication Are Corrected by the DNA Polymerase EnzymeGenomic DNA is synthesized in...
Lagging Strand Synthesis01:59

Lagging Strand Synthesis

During replication, the complementary strands in double-stranded DNA are synthesized at different rates. Replication first begins on the leading strand. Replication starts later, occurs more slowly, and proceeds discontinuously on the lagging strand.
There are several major differences between synthesis of the leading strand and synthesis of the lagging strand. 1) Leading strand synthesis happens in the direction of replication fork opening, whereas lagging strand synthesis happens in the...
Bacterial RNA Polymerase00:43

Bacterial RNA Polymerase

Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
Bacterial RNA Polymerase00:43

Bacterial RNA Polymerase

Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...

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Video Experimental Relacionado

Updated: Jun 20, 2026

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis
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DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

Published on: October 6, 2017

Evolucionando una polimerasa para análogos de bases hidrofóbicas.

David Loakes1, José Gallego, Vitor B Pinheiro

  • 1MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, United Kingdom.

Journal of the American Chemical Society
|September 26, 2009
PubMed
Resumen

Los investigadores desarrollaron una nueva polimerasa de ADN, 5D4, utilizando la evolución dirigida para replicar de manera eficiente los análogos de bases hidrofóbicas (HBA). Este avance expande el potencial de la química del ácido nucleico y la codificación para nuevas aplicaciones.

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Last Updated: Jun 20, 2026

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis
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Área de la Ciencia:

  • La bioquímica es la bioquímica.
  • Biología Molecular Biología Molecular
  • Biología sintética Biología sintética.

Sus antecedentes:

  • Los análogos de bases hidrofóbicas (HBA) ofrecen un potencial químico y de codificación ampliado para los ácidos nucleicos.
  • Sin embargo, los HBA son típicamente sustratos pobres para las polimerasas de ADN, lo que dificulta su aplicación.
  • El descubrimiento de HBA con propiedades de sustrato favorables ha sido un desafío significativo.

Objetivo del estudio:

  • Desarrollar una estrategia para mejorar las propiedades del sustrato HBA mediante la evolución dirigida de una ADN polimerasa.
  • Para utilizar la autorreplicación compartimentada (CSR) para la selección de las polimerasas capaces de replicar HBAs específicos.
  • Para identificar una polimerasa con mayor capacidad para utilizar una gama de HBAs.

Principales métodos:

  • Evolución dirigida de las polimerasas quiméricas de ADN derivadas del género Thermus.
  • La autorreplicación compartimentada (CSR) utilizando el 5-nitroindole (d5NI) y el 5-nitroindole-3-carboxamida (d5NIC) como sustratos de selección.
  • Caracterización de la actividad de la polimerasa, la especificidad del sustrato y la fidelidad utilizando ensayos bioquímicos y espectroscopia de RMN.

Principales resultados:

  • Se aisló una nueva ADN polimerasa, 5D4, con una capacidad ampliamente mejorada para utilizar HBAs.
  • 5D4 eficientemente formado y extendido d5NI y d5NIC autopares y heteropares con todas las bases estándar.
  • La polimerasa 5D4 demostró actividad con varios pares de HBA, eludió diversos HBA y permitió la amplificación por PCR de los primers que contienen HBA con alta fidelidad.

Conclusiones:

  • El enfoque de evolución dirigida produjo con éxito una polimerasa (5D4) con una utilización significativamente mejorada de HBA.
  • 5D4 amplía el repertorio de análogos de nucleobases susceptibles a la replicación y la síntesis de ADN.
  • Esta polimerasa de ingeniería es prometedora para la creación de polímeros de ácido nucleico con mayor diversidad química y funcional.