Fig 1: MCM5 UFMylation at K583 promotes origin firing.(A, B) Quantification of the inter-origin distances in HeLa cells transfected with the indicated siRNAs and plasmids. DKM 2-93 treated cells were pre-treated with DKM 2-93 (100 μM) for 1 h prior to the labeling process, and the drug was maintained throughout both CldU and IdU labeling periods. Representative fiber images are shown in (A), and quantitative data are shown in (B). Green tracts, CldU; red tracts, IdU; dashed line: replication origins. Data are presented as mean ± SD. n DNA fiber number. P value was calculated by Ordinary one-way ANOVA (****P < 0.0001; ns P > 0.05, no significance). P values: siNC vs siMCM5, 7.64e-016; siMCM5 vs siMCM5+WT, 6.46e-015; siMCM5 vs siMCM5+KR/siMCM5+DKM 2-93/siMCM5+WT + DKM 2-93/siMCM5+KR + DKM 2-93, 0.99/0.457/0.464/0.77. Scale bar, 5 μm. (C, D) Quantification of the IdU track length in the cells used in (A). Individual dots represent the length of each replication fork. Data are presented as mean ± SD. n DNA fiber number. P value was calculated by Ordinary one-way ANOVA (****P < 0.0001; ns P > 0.05, no significance). P values: siNC vs siMCM5, 1.51e-085; siMCM5 vs siMCM5+WT, 1.58e-043; siMCM5 vs siMCM5+KR/siMCM5+DKM 2-93/siMCM5+WT + DKM 2-93/siMCM5+KR + DKM 2-93, 0.983/0.811/0.999/0.997. Scale bar, 5 μm. (E) ChIP analysis to analyze the association of FLAG-CDC45 and FLAG-GINS3 at the LMNB2 origin in siRNA-treated HeLa cells transfected with the indicated plasmids. The data are presented as mean ± SD of three biological replicates (n = 3). P value was calculated by Ordinary one-way ANOVA (*P < 0.05; **P < 0.01; ****P < 0.0001). P values of FLAG-GINS3 group: shNC vs shMCM5, 6.74e-003; shMCM5 vs shMCM5+WT, 0.0119; shMCM5+WT vs shMCM5+KR, 3.61e-003. P values of FLAG-CDC45 group: shNC vs shMCM5, 1.33e-005; shMCM5 vs shMCM5+WT, 1.31e-006; shMCM5+WT vs shMCM5+KR, 1.17e-006. (F) ChIP analysis to analyze the association of endogenous MCM3 and MCM7 at the LMNB2 origin in shRNA-treated HeLa cells transfected with the indicated plasmids. The data are presented as mean ± SD of three biological replicates (n = 3). P value was calculated by Ordinary one-way ANOVA (*P < 0.05; ***P < 0.001; ****P < 0.0001; ns P > 0.05, no significance). P values of MCM3 group: shNC vs shMCM5, 1.10e-004; shMCM5 vs shMCM5+WT, 2.75e-007; shMCM5+WT vs shMCM5+KR, 0.646. P values of MCM7 group: shNC vs shMCM5, 0.0245; shMCM5 vs shMCM5+WT, 2.94e-005; shMCM5+WT vs shMCM5+KR, 0.294. Source data are available online for this figure.