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Translesion DNA Polymerases

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Fixing Double-strand Breaks

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Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Published on: September 11, 2022

Transient dsDNA breaks during pre-replication complex assembly.

Emmanouil Rampakakis¹, Maria Zannis-Hadjopoulos

¹Goodman Cancer Center and Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3A 1A3.

Nucleic Acids Research

|July 30, 2009

Summary

This summary is machine-generated.

DNA topoisomerase II (topo II) activity creates DNA breaks essential for initiating DNA replication. This process involves the DNA repair protein Ku and is crucial for pre-replicative complex assembly and cell cycle progression.

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Visualizing Single-Stranded DNA Foci in the G1 Phase of the Cell Cycle

Visualizing Single-Stranded DNA Foci in the G1 Phase of the Cell Cycle

Published on: December 22, 2023

Area of Science:

Molecular Biology
Cell Biology
Genetics

Background:

DNA replication initiation requires the assembly of the pre-replicative complex (pre-RC) during the G(1) phase.
DNA topoisomerase II (topo II) has been previously observed to associate with DNA replication origins in a cell-cycle-dependent manner.

Purpose of the Study:

To investigate the role of DNA topoisomerase II (topo II) in the activation of DNA replication origins.
To elucidate the mechanism by which topo II influences pre-replicative complex (pre-RC) assembly and G(1) phase progression.

Main Methods:

Investigated DNA topoisomerase II (topo II) activity at early-firing (lamin B2) and late-firing (hOrs8) human replication origins.
Examined the association of topo IIbeta with the DNA repair protein Ku and the pre-RC region in vivo and in vitro.
Assessed the impact of topo II inhibition on pre-RC assembly and G(1) phase duration.

Main Results:

Activation of both early and late replication origins involves transient, site-specific double-strand DNA breaks (dsDNA breaks) dependent on DNA topoisomerase II (topo II).
Topo IIbeta, in complex with the DNA repair protein Ku, introduces dsDNA breaks in a biphasic manner during early and mid-G(1) phase.
Inhibition of topo II activity disrupts pre-RC assembly, leading to a prolonged G(1) phase.

Conclusions:

Mechanistically links DNA topoisomerase IIbeta-dependent dsDNA breaks and DNA repair machinery components to the initiation of DNA replication.
Suggests a critical role for DNA topology, mediated by topo II, in the activation of replication origins.
Highlights the involvement of DNA repair proteins in the DNA replication process.