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Translesion DNA Polymerases

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...

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Related Experiment Video

Updated: Jul 2, 2025

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Published on: November 5, 2012

Studying Translesion DNA Synthesis Using Xenopus In Vitro Systems.

Antoine Aze¹, James R A Hutchins¹, Domenico Maiorano²

¹Genome Surveillance and Stability Laboratory, Institute of Human Genetics, UMR9002, CNRS-University of Montpellier, Montpellier, France.

Methods in Molecular Biology (Clifton, N.J.)

|February 23, 2024

Summary

This summary is machine-generated.

Xenopus egg extracts enable study of translesion DNA synthesis (TLS), a pathway tolerating DNA damage. This system recapitulates in vivo TLS and offers protocols for detecting TLS factors and mutagenesis.

Keywords:

Cell-free extract Chromatin DNA damage tolerance DNA replication Mutagenesis Proteomics Xenopus laevis

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Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

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Published on: November 5, 2012

Xenopus laevis as a Model to Identify Translation Impairment

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Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Published on: February 9, 2015

Area of Science:

Molecular Biology
Genetics
Biochemistry

Background:

Cell-free extracts from Xenopus eggs are valuable tools for studying fundamental cellular processes.
These processes include DNA synthesis, DNA damage response, and genome integrity maintenance.

Purpose of the Study:

To investigate translesion DNA synthesis (TLS), a critical DNA damage tolerance pathway.
To establish and utilize Xenopus cell-free extracts for studying TLS mechanisms.

Main Methods:

Utilizing Xenopus egg extracts to create cell-free assays.
Detecting chromatin-bound TLS factors via western blotting and immunofluorescence microscopy after UV irradiation.
Monitoring TLS-dependent mutagenesis.
Performing proteomic screening to identify TLS components.

Main Results:

Successfully recapitulated in vivo TLS activities within the cell-free system.
Developed protocols for analyzing TLS factor recruitment and function.
Established methods for assessing TLS-associated mutagenesis.

Conclusions:

Xenopus cell-free extracts provide a robust system for dissecting TLS pathways.
This system facilitates the study of DNA damage tolerance mechanisms relevant to embryogenesis.
The described protocols enable comprehensive analysis of TLS factors and their functional consequences.