The Enzyme-Modified Neutral Comet (EMNC) Assay for Complex DNA Damage Detection

Abstract

The comet assay is a versatile, simple, and sensitive gel electrophoresis–based method that can be used to measure and accurately quantify DNA damage, particularly single and double DNA strand breaks, in single cells. While generally this is used to measure variation in DNA strand break levels and repair capacity within a population of cells, the technique has more recently been adapted and evolved into more complex analysis and detection of specific DNA lesions, such as oxidized purines and pyrimidines, achieved through the utilization of damage-specific DNA repair enzymes following cell lysis. Here, we detail a version of the enzyme-modified neutral comet (EMNC) assay for the specific detection of complex DNA damage (CDD), defined as two or more DNA damage lesions within 1–2 helical turns of the DNA. CDD induction is specifically relevant to ionizing radiation (IR), particularly of increasing linear energy transfer (LET), and is known to contribute to the cell-killing effects of IR due to the difficult nature of its repair. Consequently, the EMNC assay reveals important details regarding the extent and complexity of DNA damage induced by IR, but also has potential for the study of other genotoxic agents that may induce CDD.

USP9X Is Required to Maintain Cell Survival in Response to High-LET Radiation

“…Given our demonstration that USP9X is required for maintaining cell survival specifically in response to relatively high-LET protons, we sought to define its mechanism of action. In the first instance, and given the propensity for high-LET radiation to cause increases in the levels of CDD, we utilized an enzyme-modified neutral comet assay ( 34 ) to monitor the levels and kinetics of repair of CDD. This assay also has the added advantage of revealing the levels and repair of DNA DSBs (in the absence of modifying enzymes).…”

“…Detection of CDD was achieved using enzyme treatment of DNA originally described by Sutherland et al. ( 32 , 33 ), but which has been employed in development of the enzyme-modified neutral comet assay, as recently described ( 34 ). In brief, cells were trypsinized, diluted to 1 × 10 5 cells/ml and 250-µl aliquots of the cell suspension placed into the wells of a 24-well plate, which was placed on ice.…”

“…In the first instance, and given the propensity for high-LET radiation to cause increases in the levels of CDD, we utilized an enzyme-modified neutral comet assay (34) to monitor the levels and kinetics of repair of CDD. This assay also has the added advantage of revealing the levels and repair of DNA DSBs (in the absence of modifying enzymes).…”

Effect of Gamma Irradiation on Viability, Pathogenicity, Reproductivity of Heterorhabditis bacteriophora and H. indica and detection of DNA Damage

“…Single cell gel electrophoresis (SCGE) or Comet assay technique has become one of the accurate methods that used for measurement of DNA damage due to its simplicity, reliability, sensitivity, low cost, and versatility (Fabbrizi et al 2021;Cordelli et al, 2021). For the first time, comet assay was able to detect the fraction of radiobiologically within an irradiated population of cells; it was using to asses that the rejoining rate of DNA breaks were relatively homogenous (Olive, 2009).…”

Impact of Gamma Irradiation on Tomato, and Pepper Growth Parameters, Phytochemical, Nematode Infectivity and detection of DNA Damage by Comet Assay

“…Gamma irradiation could affect physiology, biochemistry and morphology of the plant growth, and the cell contents of some essential compounds such as chlorophyll A, B, carotenoids, and phenolic compound, flavonoid, alkaloid and antioxidant enzyme activity (Kim et al, 2006;Rahimi and Bahrani, 2011), Moreover, the low doses may induce some useful components in plant cells (Mohajer et al, 2014), while the high doses resulting unfavorable compounds (Malencic et al, 2012;Kumar et al, 2013 andHong et al, 2018). Over the past decade, comet assay has turn into the standard methods for measurement of DNA damage due to its simplicity, reliability, sensitivity, low cost, and versatility (Fabbrizi et al 2021;Cordelli et al 2021). This assay can be used for quarantine applications to control the trade of oranges exposed to electron beam, X-rays and gamma rays, (Yunhee et al, 2018).…”