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Related Concept Videos

Base Excision Repair01:54

Base Excision Repair

One of the common DNA damages is the chemical alteration of single bases by alkylation, oxidation, or deamination. The altered bases cause mispairing and strand breakage during replication. This type of damage causes minimal change to the DNA double helix structure and can be repaired by the base excision repair (BER) pathways. BER corrects damaged DNA sequences by removing the damaged base and restoring the original base sequence using the complementary strand as a template.
The first step of...
Base Excision Repair01:54

Base Excision Repair

One of the common DNA damages is the chemical alteration of single bases by alkylation, oxidation, or deamination. The altered bases cause mispairing and strand breakage during replication. This type of damage causes minimal change to the DNA double helix structure and can be repaired by the base excision repair (BER) pathways. BER corrects damaged DNA sequences by removing the damaged base and restoring the original base sequence using the complementary strand as a template.
The first step of...
Long-patch Base Excision Repair01:02

Long-patch Base Excision Repair

Since the discovery of the two BER pathways, there has been a debate about how a cell chooses one pathway over the other and the factors determining this selection. Numerous in vitro experiments have pointed out multiple determinants for the sub-pathway selection. These are:
Nucleotide Excision Repair01:08

Nucleotide Excision Repair

Overview
Nucleotide Excision Repair01:38

Nucleotide Excision Repair

DNA Distortion and Damage
Cells are regularly exposed to mutagens—factors in the environment that can damage DNA and generate mutations. UV radiation is one of the most common mutagens and is estimated to introduce a significant number of changes in DNA. These include bends or kinks in the structure, which can block DNA replication or transcription. If these errors are not fixed, the damage can cause mutations, which in turn can result in cancer or disease depending on which sequences are...
Nucleotide Excision Repair01:08

Nucleotide Excision Repair

Overview

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Related Experiment Video

Updated: May 19, 2026

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons
15:01

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

Published on: August 6, 2012

In vitro base excision repair using mammalian cell extracts.

Jason L Parsons1, Grigory L Dianov

  • 1Gray Institute for Radiation, Oncology and Biology, University of Oxford, Oxford, UK.

Methods in Molecular Biology (Clifton, N.J.)
|September 4, 2012
PubMed
Summary

This study describes a method for analyzing base excision repair (BER) in mammalian cell extracts in vitro. By using DNA substrates with specific lesions and analyzing the results with denaturing gels and phosphor imaging, the researchers show that mammalian extracts can process DNA damage. The method allows for a detailed look at how BER functions in a system that mimics cellular conditions. This approach may help researchers better understand DNA repair mechanisms and their role in diseases like cancer.

Keywords:
base excision repairDNA repair mechanismsmammalian cell extractsin vitro DNA repair

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Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons
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Area of Science:

  • Molecular biology techniques in DNA repair
  • Cellular response to mutagens within biochemistry
  • Genome stability research in medical genetics

Background:

DNA damage occurs naturally through metabolic processes and can also result from exposure to external agents. Base excision repair (BER) is a key pathway for correcting small DNA lesions. Prior research has shown that BER is vital for preventing mutations and maintaining genomic integrity. However, the precise mechanisms and interactions of BER components remain partially understood. This gap motivated the need for in vitro models that can simulate BER in controlled environments. No prior work had resolved how mammalian cell extracts can be systematically used to study BER. Understanding BER is essential for addressing diseases linked to DNA repair deficiencies. This paper contributes by offering a detailed in vitro BER protocol using mammalian extracts.

Purpose Of The Study:

The aim of this study is to provide a method for analyzing BER in vitro using mammalian cell extracts. This approach allows for the investigation of BER mechanisms in a controlled setting. The specific problem addressed is the lack of standardized protocols for BER assays using mammalian systems. The motivation stems from the need to understand how BER functions in higher organisms. This method enables the study of BER on both oligonucleotide and circular DNA substrates. The study also seeks to clarify how DNA lesions are processed in mammalian cells. By using denaturing acrylamide gel electrophoresis, the researchers aim to visualize repair products. This work may help clarify the molecular basis of DNA repair-related diseases.

Main Methods:

The researchers used mammalian cell extracts to perform BER assays in vitro. They prepared DNA substrates containing site-specific lesions using oligonucleotides and closed circular DNA. These substrates were incubated with cell extracts to allow repair processes to occur. The reaction products were then analyzed using denaturing acrylamide gel electrophoresis. Phosphor imaging was employed to detect and quantify the repair outcomes. The method includes steps for extract preparation and substrate modification. The use of both oligonucleotide and circular DNA allows for a comprehensive analysis of BER. This approach enables the study of BER in a system that mimics cellular conditions.

Main Results:

The in vitro BER assay successfully detected repair activity in mammalian cell extracts. Both oligonucleotide and circular DNA substrates showed evidence of lesion processing. Denaturing gel electrophoresis revealed distinct repair products, indicating active BER. Phosphor imaging confirmed the presence of repaired DNA fragments. The results suggest that mammalian extracts can efficiently process site-specific DNA lesions. The method is effective for analyzing BER in a controlled setting. The use of closed circular DNA substrates provided insights into BER in genomic contexts. These findings support the utility of this assay for studying DNA repair mechanisms.

Conclusions:

The study demonstrates that mammalian cell extracts can be used effectively in in vitro BER assays. The method allows for the analysis of BER on both oligonucleotide and circular DNA substrates. The results suggest that BER can be studied in a system that closely mimics cellular conditions. The use of denaturing gel electrophoresis and phosphor imaging provides clear evidence of repair activity. The findings support the potential of this assay for investigating DNA repair mechanisms. The study does not propose generalizations beyond the observed results. The method may help clarify the role of BER in DNA repair-related diseases. The authors do not suggest future directions or drug targets based on these findings.

The assay tests the ability of mammalian cell extracts to process site-specific DNA lesions through base excision repair (BER).

Oligonucleotides allow for precise lesion placement, while circular DNA mimics genomic DNA structures in BER studies.

Phosphor imaging detects and quantifies DNA repair products, providing a visual and quantitative readout of BER activity.

Denaturing gels separate DNA fragments based on size and charge, allowing researchers to identify repair products.

The substrates contained site-specific DNA lesions, including base lesions and apurinic (AP) sites.

The authors suggest that this method can help study BER mechanisms and their relevance to DNA repair-related diseases.