Time course curves of UV-irradiated HCT116(wt), HCT116(Rad18^−/−), and HCT116(RAD18^−/−/+RAD18), which is a HCT116(Rad18^−/−) cell line expressing RAD18 for complementation.

<p>Cells were BrdU pulse labelled and mock treated or irradiated with UV (20 J/m²) and replication fork progression was followed by alkaline BrdU comet PRR assay. The % comet tail DNA, as a measure of discontinuity was monitored in untreated and in UV treated cells. Each data point represents the mean of three independent experiments. Error bars indicate standard deviations.</p

Main principle of the alkaline BrdU comet PRR assay.

<p>(<b>A</b>) Experimental setup. (<b>B</b>) Chematic illustration of neighbouring origins and bidirectional way of replication fork movement. BrdU incorporates into newly replicated DNA strands during short pulse labelling. As replication progresses, the labelled strands move apart from the replication fork during the chase period and are incorporated into high molecular weight DNA. In the untreated cells (-UV) the discontinuities of DNA strands are abolished gradually by ligation of DNA strands elongated from neighbouring origins in a process called DNA maturation. In the presence of DNA adducts caused by UV irradiation (+UV), replication fork movement is inhibited and the nascent DNA remains discontinuous for a longer time. (<b>C</b>) At early stages of replication of an undamaged template, the short stretches of newly replicated DNA can be separated from the template strand by alkaline unwinding and visualised as comet tail DNA after short alkaline electrophoresis followed by immunostaining with anti-BrdU antibody. Two different types of events are presented as examples with neighbouring replication origins: labelling of new origin firing and an elongating fork. As the replication fork elongates on undamaged template and DNA maturation is completed by the ligation step, the DNA becomes a continuous, high molecular weight molecule, forming the comet head. On damaged templates DNA replication is inhibited and the DNA remains fragmented, the labelled fragments migrate into the comet tail and can be detected as low molecular weight fragments even at late time points. Examples of detectable labelled fragments are shown as (a) termination of a new origin firing, (b) termination of elongation and (c) inhibition of elongation.</p

Dose dependent inhibition of replication progression caused by UV as detected by BrdU comet PRR assay.

<p>Cells were BrdU pulse labelled and mock treated or irradiated with UV and replication fork progression was followed by alkaline BrdU comet PRR assay. BrdU was detected by immunostaining, and quantitative measurement of comet tail DNA was done by Komet5 software. The % comet tail DNA, as a measure of discontinuity was illustrated. (<b>A</b>) Dose-effect curve of HeLa cells after 4 h recovery time. (<b>B</b>) Dose-effect curve of HCT116(wt), HCT116(Rad18^−/−)cell lines after 6 hour recovery. Each data point represents the mean of three independent experiments. Error bars indicate standard deviations. (<b>C</b>) Representative alkaline BrdU comet PRR images as revealed by anti-BrdU immunostaining of HCT116(wt) cells irradiated with increased UV dose as indicated followed by 6 hour recovery. (<b>D</b>) As (C) but HCT116(Rad18^−/−) cells were used instead of wild type cells.</p

Alkaline BrdU comet PRR images of HCT116(RAD18^−/−) cells after UV irradiation.

<p>(<b>A</b>) UV irradiated (20 J/m²) cells were allowed to recover for 6 hour. Representative images of UV treated cells display highly discontinuous, fragmented tails. The modified BrdU comet assay allowed proper differentiation between comet head and tail, which is essential for precise quantitation. Following the BrdU comet assay, the incorporated BrdU was identified by anti-BrdU primary antibody and cells were detected using Alexa Fluor 488 conjugated secondary antibody (green, panel I). Ethidium bromide counterstaining of the same cells (red, panel II) and merged images (yellow, panel III) are shown. (<b>B</b>) BrdU immunostaining detects S-phase cells without synchronisation. HCT116(RAD18^−/−) cell were UV irradiated (40 J/m²) and allowed to recover for 6 hour. Staining was carried out as described in (A). Arrows indicate the non-S-phase cells which has not been stained with anti-BrdU antibody but only with ethidium bromide.</p

Alkaline BrdU comet PRR assay images of untreated and UV-irradiated HeLa cells representing the progression of replication as detailed in Figure 1.

<p>The cells were pulse labelled with BrdU before irradiation with 20 J/m² UV-C or mock treatment. After the indicated time (0–6 h) single-stranded DNA fragments were separated from matured DNA by alkaline single cell electrophoresis followed by immunostaining using fluorescent anti-BrdU antibody (green) or staining with ethidium-bromide (red). The much higher sensitivity of the BrdU comet assay as compared to the basic comet assay was demonstrated by showing the anti-BrdU stained and ethidium bromide counterstained images of the same cells.</p