Abstract
A new method of the light microscopy detection of BrdU-labeled DNA in situ is described. It is based on the oxidative attack at the deoxyribose moiety by copper(I) in the presence of oxygen, which leads to the abstraction of hydrogen atom from deoxyribose culminating in the elimination of the nucleobase, scission of the nucleic-acid strand and formation of frequent gaps. The gaps allow the reaction of the antibodies with the commonly used markers of replication (e.g. 5-bromo-2′-deoxyuridine), which are otherwise masked. The method developed makes it possible to detect nuclear and mitochondrial DNA replication efficiently. In most cases, it does not inhibit effective protein detections and in addition enables simultaneous localization of newly-synthesized RNA. The alternative presently-used methods result in protein denaturation and/or extensive DNA cleavage followed by the DNA-bound proteins peeling off.
Highlights
5-Bromo-29-deoxyuridine (BrdU) or its alternatives, 5-chloro-29deoxyuridine (CldU) and 29-deoxy-5-iodouridine (IdU), are widely used for the visualization of sites containing newly replicated DNA
The system of oxidative attack at the deoxyribose moiety by copper or iron ions, sodium ascorbate and hydrogen peroxide for isolated DNA as an effective DNA cleavage system was described earlier [16], and the gaps created by the oxidative attack at the deoxyribose moiety can theoretically make incorporated 5-halo-nucleosides accessible, it was not clear how effective the formation of gaps in DNA is in fixed cells, what is the sensitivity of this approach, and what the effect of the conditions used is on the cellular structure
The system based on copper ions was chosen in this study as the experiments performed so far have shown that the system based on copper ions, sodium ascorbate and hydrogen peroxide is more efficient than the system based on iron ions [24]
Summary
5-Bromo-29-deoxyuridine (BrdU) or its alternatives, 5-chloro-29deoxyuridine (CldU) and 29-deoxy-5-iodouridine (IdU), are widely used for the visualization of sites containing newly replicated DNA. A technique based on the reaction of 29-deoxy-5ethynyluridine (EdU) that bypasses the necessity to use antibodies owing to the possibility of the reaction of the terminal alkyne of EdU with the azido group attached to the fluorescent dye has been developed [7], it suffers from the fact that EdU can exhibit cytotoxicity with an unknown mechanism that varies for different cell lines [8,9]. It seems that the sensitivity of the microscopic detection of the mitochondrial replication by EdU is lower than by BrdU. For validation of the method developed, we have compared it with the methods presently used for the detection of replicated DNA by means of 5-halonucleosides
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