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Review
. 2018 Nov 17;23(11):3007.
doi: 10.3390/molecules23113007.

DNA Replication: From Radioisotopes to Click Chemistry

Anna Ligasová  1 , Karel Koberna  2
Affiliations
Review

DNA Replication: From Radioisotopes to Click Chemistry

Anna Ligasová et al. Molecules. .

Abstract

The replication of nuclear and mitochondrial DNA are basic processes assuring the doubling of the genetic information of eukaryotic cells. In research of the basic principles of DNA replication, and also in the studies focused on the cell cycle, an important role is played by artificially-prepared nucleoside and nucleotide analogues that serve as markers of newly synthesized DNA. These analogues are incorporated into the DNA during DNA replication, and are subsequently visualized. Several methods are used for their detection, including the highly popular click chemistry. This review aims to provide the readers with basic information about the various possibilities of the detection of replication activity using nucleoside and nucleotide analogues, and to show the strengths and weaknesses of those different detection systems, including click chemistry for microscopic studies.

Keywords: click chemistry; indirect immunocytochemistry; isotopes; nucleoside and nucleotide analogues.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
The detection of BrdU using DNase I and exonuclease III. Examples of microscopic images of cell nuclei labeled for 5 min with BrdU and fixed with formaldehyde. The DNA was labeled by DAPI. The scale bar—50 μm. Adapted with permission from Ligasová et al., 2017, Looking for ugly ducklings: The role of the stability of the BrdU-antibody complex and the improved method of the detection of DNA replication. PLoS ONE 12(3): e0174893 [47].
Figure 2
Figure 2
Labeling patterns of newly replicated DNA visualized on DNA fibers. DNA was first labeled with CldU for 10 min (green) and then with IdU for 5 min (red). Two isolated elongating forks (arrow) and replicons with initiation and termination sites (arrowheads) are shown. Bar = 5 µm. Adapted with permission from Malínský et al., 2001, The supply of exogenous deoxyribonucleotides accelerates the speed of the replication fork in the early S phase. Journal of Cell Sciences 114, 747–750 [54].
Figure 3
Figure 3
Three replication patterns visualized by BrdU immuno-detection during the S phase. The HeLa cells were labeled with BrdU for 10 min, fixed (2% formaldehyde, 10 min), permeabilized (0.2% Triton X-100, 10 min), and treated with 4N HCl (20 min). The incorporated BrdU was visualized using anti-BrdU antibody and Cy3 labeled anti-mouse antibody. Type I—large number of small replication sites characteristic for early S phase; type II—replication sites concentrated at the periphery of the nucleus and nucleoli (mid S phase); type III—large replication sites over heterochromatin (late S phase).
Figure 4
Figure 4
The detection of the BrdU-labeled DNA in mitochondria. The detection of the BrdU-labeled DNA (red in the color image) after a 1-hour BrdU pulse in cells treated with 4 mM copper(I) for 60 s followed by exonuclease III cleavage is shown. A simultaneous co-localization with the mitochondrial marker MTC02 has been performed (green in the color image). Bar: 10 µm. Adapted with permission from Ligasová et al., 2012, Atomic Scissors: a new method of tracking the 5-bromo-2′-deoxyuridine-labeled DNA in situ. PLoS ONE 7(12): e52584 [42].
Figure 5
Figure 5
Labeling of DNA using EdU. NIH 3T3 cells labeled by incubation overnight with 2 μM of EdU were fixed and reacted successively with 10 μM of Alexa488-azide and 10 μM of Alexa594-azide, respectively. The cells were imaged by fluorescence microscopy. (Left) Alexa488-azide stain. (Center) Alexa594-azide stain. (Right) Overlay of the Alexa488 and Alexa594 images. Adapted with permission from Salic, A., Mitchison, T.J., 2008, A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci U S A 105, 2415–2420, Copyright (2008) National Academy of Sciences U.S.A [76].
Figure 6
Figure 6
The use of EdU to label DNA in cells. (a) Structure of EdU. (b) Scheme of the click reaction for detecting EdU incorporated into cellular DNA. The terminal alkyne group, exposed in the major groove of the DNA helix, readily reacts with an organic azide (R can be any fluorophore, hapten, electron-dense particle, quantum dot, etc.) in the presence of catalytic amounts of Cu(I). Adapted with permission from Salic, A., Mitchison, T.J., 2008, A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci U S A 105, 2415–2420, Copyright (2008) National Academy of Sciences U.S.A. [76].
Figure 7
Figure 7
The detection of EdU in fixed HeLa cells and the simultaneous localization of BrdU and EdU. (a) The picture shows examples of the detection of EdU in fixed and permeabilized cells with six anti-BrdU antibodies after a ten-minute EdU labeling pulse. Note that only the clone MoBu-1 exhibits no signal. Bar: 20 µm. (b) The results of simultaneous localization of BrdU and EdU of cells labeled for 5 min with BrdU and then after 13 h for 20 min with EdU is shown. The upper set of images represents the detection of both signals without the application of the blocking step by means of 2 mM of azidomethylphenylsulfide. The bottom set of images represents the detection of both signals with the application of the blocking step by means of 2 mM of azidomethylphenylsulfide. Bar: 20 µm. Adapted with permission from Liboska et al., 2012, PLoS ONE 7(12): e51679 [100].
Figure 8
Figure 8
An example of labeling of DNA replication using biotin tagged deoxyuridine triphosphate. HeLa cells were hypotonically treated according to [119,121], fixed (2% formaldehyde, 10 min) and permeabilized (0.2% Triton X-100, 10 min). The biotin was detected using an anti-biotin primary antibody followed by the Alexa Fluor 488 anti-rabbit antibody (green in the color figure). The DNA was stained by DAPI (blue in the color figure). Bar: 50 µm.
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