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Replication in Eukaryotes

Replication in Eukaryotes

Replication in Eukaryotes

Replication in Eukaryotes

In eukaryotic cells, DNA replication is highly conserved and tightly regulated. Multiple linear chromosomes must be duplicated with high fidelity before cell division, so there are many proteins that fulfill specialized roles in the replication process. Replication occurs in three phases: initiation, elongation, and termination, and ends with two complete sets of chromosomes in the nucleus.
Many Proteins Orchestrate Replication at the Origin
Eukaryotic replication follows many of the same...

Conservative Site-specific Recombination and Phase Variation

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...

The DNA Replication Fork

The DNA Replication Fork

An organism’s genome needs to be duplicated in an efficient and error-free manner for its growth and survival. The replication fork is a Y-shaped active region where two strands of DNA are separated and replicated continuously. The coupling of DNA unzipping and complementary strand synthesis is a characteristic feature of a replication fork. Organisms with small circular DNA, such as E. coli, often have a single origin of replication; therefore, they have only two replication...

The DNA Replication Fork

The DNA Replication Fork

Proofreading

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...

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Updated: Feb 18, 2026

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

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Published on: January 8, 2015

La edición precisa en las horquillas de replicación del ADN permite la ingeniería del genoma múltiple en eucariotas

Edward M Barbieri¹, Paul Muir¹, Benjamin O Akhuetie-Oni¹

¹Department of Molecular, Cellular, & Developmental Biology, Yale University, New Haven, CT 06520, USA; Systems Biology Institute, Yale University, West Haven, CT 06516, USA.

|November 21, 2017

Resumen

Este estudio introduce un nuevo método de ingeniería del genoma múltiple en la levadura. Permite modificaciones precisas y eficientes del ADN sin rupturas de doble cadena, creando una amplia diversidad genética para la ingeniería de vías.

Palabras clave:

Replicación del ADN Rad51 (en inglés)Edición del genoma Recombinación homóloga ingeniería metabólica Ingeniería del genoma múltiple Productos naturales Oligodeoxinucleótidos del ADN ss

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Área de la Ciencia:

Biología molecular
Biología sintética
La genética

Sus antecedentes:

Los métodos actuales de ingeniería genómica a menudo se basan en rupturas de doble hebra y recombinación homóloga, lo que puede conducir a mutaciones no deseadas.
La ingeniería del genoma múltiple es crucial para crear diversidad genética compleja de manera eficiente.

Objetivo del estudio:

Desarrollar una nueva tecnología de ingeniería del genoma múltiple, eficiente y precisa en Saccharomyces cerevisiae.
Demostrar la capacidad de esta tecnología para la diversificación combinatoria de las vías biosintéticas.

Principales métodos:

Se utiliza el recocido de oligonucleótidos sintéticos en la cadena rezagada de la replicación del ADN.
Evitó la necesidad de recombinación homóloga dirigida por Rad51 y roturas de ADN de doble cadena.
Se ha logrado la incorporación simultánea de múltiples oligonucleótidos y mutaciones dirigidas.

Principales resultados:

Se han demostrado modificaciones cromosómicas precisas a resolución de un solo par de bases con una eficiencia superior al 40%.
Incorporó con éxito hasta 12 oligonucleótidos y 60 mutaciones en una sola transformación.
Diversidad genómica combinatoria generada superior a 10^5 mediante transformaciones iterativas.
Diseñaron una vía de biosíntesis de β-caroteno heteróloga, produciendo variantes con niveles alterados de carotenoides debido a mutaciones precisas.

Conclusiones:

El método desarrollado ofrece un enfoque independiente de Rad51, libre de ruptura de doble cadena para la ingeniería del genoma múltiple.
Esta tecnología permite la modificación de alta eficiencia, precisa y combinatoria de los genomas eucariotas.
La estrategia es automatizable y aplicable para generar una diversidad genómica significativa para diversas aplicaciones, incluida la ingeniería metabólica.