Name: Video: Analyzing DNA Break Induction in U2OS Cancer Cells Using Comet Assay
Uploaded: 2024-08-23T14:20:00
Description: 47 Views. Analyzing DNA Break Induction in U2OS Cancer Cells Using Comet Assay

analyzing dna break induction in u2os cancer cells using comet assay

Investigating the Extent of DNA Breaks Occurring During DNA Replication 

The comet assay is a gel electrophoresis method used to detect DNA breaks at the single-cell level. It relies on the principle that open-ended DNA breaks migrate under electrophoretic conditions, while intact DNA remains largely static. Broken DNA migrates because breaks result in the relaxation of supercoiled DNA, causing its gradual spatial relocation towards the anode in the electrophoretic chamber, which results in a comet-like appearance observed by immunofluorescence1,2. The extent of DNA damage is then measured by quantifying the amount of broken DNA that has migrated relative to the compact DNA.
The two most commonly used comet assay methods are the alkaline comet (AC) assay and the neutral comet (NC) assay. The AC assay is performed under denaturing conditions using a high alkaline pH solution, while the NC assay is carried out in a solution with neutral pH. Both NC and AC assays can reliably detect DNA breaks that occur in the nucleus. However, the alkaline version is also advantageous for detecting alkali-labile sites3. Both formats of the assay require the use of lysis conditions to ensure that DNA is free of proteins prior to electrophoresis.
This assay is a simple method for detecting DNA breaks and offers several unique advantages for determining cellular DNA damage. The method is relatively easy to set up and requires reagents that can either be prepared in the laboratory or purchased from commercial vendors. As a single-cell resolution technique, the assay requires very little starting material. Notably, the NC assay can measure breaks with high sensitivity, reported to detect between 50 and 10,000 breaks per cell4. The versatility of this technique is demonstrated by its wide range of applications, including ecotoxicology5, human biomonitoring6, and genotoxicity studies7. The assay can also be reliably used across various cell types and adapted for high-throughput assays8 and for assessing breaks at specific genomic regions9. Thus, this assay serves as a rapid and reliable technique for investigating break formation at the single-cell level.
The process of DNA replication is constantly challenged by several endogenous and exogenous stressors10,11. Stalling of replisomes due to such lesions can cause DNA breaks and result in heightened genome instability. DNA breaks also often arise as replication intermediates to facilitate DNA repair12. Additionally, several chemotherapeutic agents induce replication-dependent double-strand breaks (DSBs), such as camptothecin (CPT)13,14,15and PARP inhibitors16,17. Thus, this assay serves as a powerful methodology to study the nature of DNA lesions, assess the processing of replication forks, and investigate the therapeutic potential of DNA-damaging agents. Adaptations of the assay that involve labeling newly synthesized DNA with BrdU have been useful for studying replication-associated stress phenotypes18,19,20,21. Notably, the NC assay is a reliable method for studying DNA break induction in the context of replication, as demonstrated in studies involving the characterization of fork proteins22,23, analysis of replication intermediates24,25, and investigation of transcription-replication conflicts in genome maintenance26,27. Herein, we outline a NC assay protocol with the goal of quantifying DNA breaks during replication in human cells. This protocol can be readily implemented by researchers interested in assessing replication stress-associated damage in a wide variety of dividing human cells.

Author Spotlight: Unveiling the Role of SNF2L in Replication Fork Stability and Genome Duplication

Detection of DNA Breaks in Dividing Human Cells by Neutral Comet Assay

Analyzing DNA Break Induction in U2OS Cancer Cells Using Comet Assay

analyzing-dna-break-induction-in-u2os-cancer-cells-using-comet-assay

Research

JoVE Journal

Bioengineering

47 Views. Analyzing DNA Break Induction in U2OS Cancer Cells Using Comet Assay

Video: Analyzing DNA Break Induction in U2OS Cancer Cells Using Comet Assay

DNA replication is constantly challenged by a wide variety of endogenous and exogenous stressors that can damage DNA. Such lesions encountered during genome duplication can stall replisomes and convert replication forks into double-strand breaks. If left unrepaired, these toxic DNA breaks can trigger chromosomal rearrangements, leading to heightened genome instability and an increased likelihood of cellular transformation. Additionally, cancer cells exhibit persistent replication stress, making the targeting of replication fork vulnerabilities in tumor cells an attractive strategy for chemotherapy. A highly versatile and powerful technique to study DNA breaks during replication is the comet assay. This gel electrophoresis technique reliably detects the induction and repair of DNA breaks at the single-cell level. Herein, a protocol is outlined that allows investigators to measure the extent of DNA damage in mitotically dividing human cells using fork-stalling agents across multiple cell types. Coupling this with automated comet scoring facilitates rapid analysis and enhances the reliability in studying induction of DNA breaks.

This protocol is designed to investigate the extent of DNA damage occurring during DNA replication. Under neutral conditions, the induction of DNA breaks can be readily assessed within a short time frame. Additionally, the protocol is adaptable to other cell types and various replication stress reagents.

DNA replication is constantly challenged by a wide variety of endogenous and exogenous stressors that can damage DNA. Such lesions encountered during ...