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. 2022 May 7;50(9):5111–5128. doi: 10.1093/nar/gkac299

Figure 1.

Figure 1.

SIRT1 deacetylase activity facilitates normal S-phase progression and origin dormancy. (A) SIRT1 knock-out (KO) HCT116 cells were reconstituted with either wild-type (WT) or mutant (H363Y, lacks deacetylase activity) SIRT1. S-phase progression in HCT116 cells harboring WT, KO or H363Y SIRT1 was measured by flow cytometry using a standard EdU incorporation/DAPI assay. Cell cycle phases were gated as shown in the far-left panel and representative cell cycle profiles from each cell line are shown (A). ES-early S, MS-mid S, LS-late S and NS- non-EdU + low EdU (red dots). (B) A bar plot showing the percentage of WT, KO and H363Y cells in G1, S (ES, MS, LS) and G2/M phases, along with cells exhibiting skewed replication (low-EdU, non-EdU), >G2/M and sub-G1 phases. The data were obtained from four replicates. (C) Schematic for the experimental strategy measuring replication profile changes using DNA combing. (D) Representative images shown DNA combing images of fiber samples from WT, KO and H363Y cells without any treatment (white scale bar = 50 kb). (E and F) Replication fork progression rates (E) and replication inter-origin distances (F) in untreated WT, KO and H363Y cells in the presence or absence of Aphidicolin (Aph). Aph was added (0.8 μM) 24 h prior to CldU. The Mann–Whitney test was used to determine statistical significance. ns = non-significant, *** represents P< 0.001 and **** represents P< 0.0001. (G) Representative DNA-combing image of symmetric and asymmetric replication forks on a single DNA fiber (white scale bar = 50 kb). (H) Inactive SIRT1 (KO or H363Y) increases the frequency of asymmetric replication forks in HCT116 cells. DNA-combing analysis was performed as in Figure 1C. Lengths of replicating signals from left and right forks emanating from the same origins were compared, and replication forks were classified as asymmetric if the difference in length between the right and left fork was >30%. n = total number of forks, AF = the percentage of asymmetric forks. Chi-square test was used to determine the statistical significance between results for WT versus KO and WT versus H363Y.