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Methods for registration of spontaneous DNA instability in mammalian cells
M V Filatov1, R A Pantina, L A Noskin
1St. Petersburg Nuclear Physics Institute, Gatchina, Russian Federation. filatov@omrb.pnpi.spb.ru
Abstract:
A phenomenon of spontaneous DNA instability displays itself as the low level of repair DNA synthesis that takes place during any cell cycle phases. However, there is a problem in detection of very low intensive repair DNA synthesis. This paper suggests two approaches to detect the spontaneous DNA instability. The first method involves a blockade of the DNA gaps sealing by a combination of inhibitors, hydroxyurea and arabinofuranosyl cytosine. An accumulation of single strand gaps leads to production of DNA double strand breaks and results to reproductive inactivation of cells. It was shown that registration of both these events by different methods (such as viscoelastometry of DNA, orthogonal pulse electrophoresis or comet assay for double strand breaks as well as effectiveness of colony growth for cell inactivation) may be used as suitable measure of the spontaneous DNA instability. The second approach bases on photolysis of bromodeoxyuridine incorporated into repair DNA patches during the spontaneous repair DNA synthesis. Long wave UV irradiation of cells containing bromodeoxyuridine labeled DNA stained with Hoechst 33342 causes their inactivation. Experimental results presented confirm that both methods actually detect the spontaneous DNA instability. It takes note of the spontaneous DNA instability varies for cells from different tissues and species and increases during aging.
Insights
This study introduces two novel methods to detect spontaneous DNA instability, a key indicator of cellular aging and tissue variation. These techniques offer new ways to measure DNA repair synthesis and its consequences.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Spontaneous DNA instability is characterized by low-level DNA repair synthesis across cell cycle phases.
- Detecting this low-intensity repair DNA synthesis poses a significant challenge in current methodologies.
Purpose of the Study:
- To develop and validate two distinct approaches for detecting spontaneous DNA instability.
- To establish reliable measures for assessing DNA repair synthesis and its cellular impact.
Main Methods:
- Method 1: Inhibiting DNA gap sealing with hydroxyurea and arabinofuranosyl cytosine, followed by measuring DNA double-strand breaks and cell inactivation.
- Method 2: Incorporating bromodeoxyuridine during repair synthesis and inducing cell inactivation via long-wave UV photolysis.
Main Results:
- Both developed methods successfully detect spontaneous DNA instability.
- The study demonstrates that spontaneous DNA instability varies across different tissues and species.
- A notable increase in spontaneous DNA instability was observed during cellular aging.
Conclusions:
- The proposed methods provide effective means to quantify spontaneous DNA instability.
- Spontaneous DNA instability is a dynamic process influenced by tissue type, species, and aging.