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. 2011 Feb 9;39(9):e60. doi: 10.1093/nar/gkr063

Figure 3.

Figure 3.

Effects of EdU on the kinetics of DNA replication. (A) Scheme of experiment. adh1:tk adh1:hENT1 (P2470) cells were arrested in G1 by nitrogen starvation by growing in EMM lacking nitrogen for 16 h at 26°C, then released from the block at 32°C in EMM containing nitrogen, in the presence (1 μM) or absence of EdU, (B) Cells from the −EdU culture were processed for analysis of DNA content after SYTOX Green staining by flow cytometry in the absence of conjugation to fluorescent azide, (C) Cells from the +EdU culture were conjugated to fluorescent azide (Alexa Fluor 488) and fluorescence was quantitated by flow cytometry, (D) Cells from the +EdU culture were processed for analysis of DNA content after SYTOX Green staining by flow cytometry in the absence of conjugation to fluorescent azide. Cells arrested in G1 by nitrogen starvation (i.e. t = 0 time point) are mainly in G1 (1C), but 15–20% cells are in G2 (2C) (17); these cells have not replicated their DNA at the 5 h time point, and this shows as a shoulder (arrow) on the main 2C peak generated by replication of 1C cells. This peak is shifted to the left of the 2C position defined by the ‘G2 minus-nitrogen population’, probably due to some effect of EdU incorporation on SYTOX Green fluorescence (see text for further discussion). A longer time course shows disappearance of the shoulder at 6–7 h (Supplementary Figure S3) consistent with an earlier study showing that cells in the ‘G2 minus-nitrogen population’ replicate their DNA around this time (17), (E) Analysis of chromosomes by PFGE. DNA plugs were prepared from cells released from a G1 arrest as in (A) and grown without (lanes 1–3, ‘no treatment’) or with 10 μM EdU (lanes 4, 5, ‘+EdU’) for the times shown. As a control for partially replicated chromosomes, cells were grown in the presence of 12 mM HU (lanes 6–7 ‘+HU’). ‘[’indicates a smear in the 5 h +EdU sample, suggesting fragmented DNA. Equal quantities of cells were processed for each well. (F) Flow cytometric analysis of cells used to prepare samples shown in (E), showing that the HU block to DNA replication is maintained at the 5 h time point.