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Published on: September 5, 2017
Determination of UV-induced DNA damages to suppress protein expression using reporter gene assay-based single cell
Yu Kyung Tak1, Won Young Kim, Enyoung Han
1Research Institute of Pharmaceutical Sciences and College of Pharmacy, Seoul National University, Seoul 151-742, South Korea.
Abstract:
Effects of UVB or UVC-induced DNA damage were simultaneously monitored at single cellular level by analyzing the change of yellow fluorescent protein (YFP) and red fluorescent protein (RFP) expression in human embryonic kidney (HEK) 293 cells using multicolor single-cell imaging cytometry. The method is based on the idea that there exists a quantitative correlation between the degree of UV-induced DNA damage and protein expression. A cotransfection assay was performed using UVB irradiated YFP and UVC irradiated RFP genes to eliminate cell-to-cell variation in protein expression yield. Up to an UVB irradiation dose of 50kJ/m(2), YFP expression yield did not change compared to control. On the other hand, RFP expression yield decreased remarkably as the UVC dose increased from 79.5 to 159J/m(2). The results showed that a certain level of DNA damage is efficiently repaired by intracellular repair mechanism and does not influence protein mutation. In addition, it was found that the amount of DNA damage induced by UVB in sunlight would not interfere with normal protein expression in the human body. Single-cell imaging cytometry is a cell lysis-free approach to directly monitor the intracellular correlation between the degree of UV-induced DNA damage and protein expression.
Insights
UV radiation can damage DNA, but cells can repair it. This study shows that moderate UVB exposure does not affect protein expression, while higher UVC doses do, using advanced single-cell imaging.
Area of Science:
- Molecular Biology
- Cell Biology
- Biophysics
Background:
- Ultraviolet (UV) radiation induces DNA damage, potentially affecting cellular functions.
- Understanding the cellular response to UV damage is crucial for human health.
- Existing methods for assessing UV-induced DNA damage can be complex and time-consuming.
Purpose of the Study:
- To develop and validate a novel method for simultaneously monitoring UV-induced DNA damage and protein expression at the single-cell level.
- To investigate the differential effects of UVB and UVC radiation on DNA damage and protein expression.
- To assess the cellular repair capacity and its impact on protein mutation.
Main Methods:
- Utilizing multicolor single-cell imaging cytometry in human embryonic kidney (HEK) 293 cells.
- Employing a cotransfection assay with yellow fluorescent protein (YFP) for UVB and red fluorescent protein (RFP) for UVC.
- Analyzing changes in YFP and RFP expression as indicators of DNA damage and repair.
Main Results:
- YFP expression remained stable up to 50 kJ/m² UVB, indicating efficient DNA repair without affecting protein yield.
- RFP expression significantly decreased with increasing UVC doses (79.5–159 J/m²), suggesting dose-dependent DNA damage.
- The study demonstrated a quantitative correlation between UV-induced DNA damage and protein expression levels.
Conclusions:
- Intracellular repair mechanisms can efficiently handle certain levels of DNA damage, preventing protein mutation.
- UVB radiation, as found in sunlight, is unlikely to interfere with normal protein expression in humans.
- Single-cell imaging cytometry offers a lysis-free approach to study the direct intracellular relationship between DNA damage and protein expression.
