Fig 2: MCM10 deficiency causes significant cell cycle and DNA synthesis defects.a Cell cycle distribution of wild type and MCM10+/- cells. The percentage of each population in G1- (green), S- (purple) or G2/M-phase (gray) is indicated. b Cell cycle distribution of wild type and MCM10+/- cell lines from three biological replicates. The average percentage of each population in G1- (green), S- (purple), and G2/M-phase (gray) is indicated. Individual data points are indicated (gray circles). c Schematic of flow cytometry analysis. Cell-cycle phase is defined by DNA content, EdU incorporation, and chromatin-loaded MCM2. d Analytical flow cytometry plots for wild type and MCM10+/- cells. G1-phase/MCM positive cells (blue), S-phase/MCM positive cells (orange) and G1- or G2/M-phase/MCM negative cells (gray) are indicated. e Comparison of origin licensing (left) and quantification of average G1 loaded MCM2 in biological replicates (n = 3) of wild type (gray) and MCM10+/- cells (blue/green). f Comparison of S-phase DNA synthesis (left) and average EdU intensity in biological replicates (n = 3) of wild type (gray) and MCM10+/- cells (red/orange). Error bars in b, e, and f indicate SD, and significance was calculated using an unpaired, two-tailed student’s t-test with *<0.05; **<0.01, ***<0.001. g Inter-origin distance (IOD) quantification in wild type (blue) and MCM10+/- cells (red). Average IOD and number (n) quantified are listed. h Fork speed in wild type (blue) and MCM10+/- cells (red). Average fork speed (kb/minute) and number (n) quantified are listed. i Fork stability in wild type (blue) and MCM10+/- cells (red). Average fork stability and number (n) quantified are listed. Statistical significance for g–i was calculated using an unpaired, two-tailed Mann-Whitney Ranked Sum Test with *<0.05; **<0.01, ***<0.001. j Chromatin associated PCNA, PCNA-Ub, and phospho-RPA32, which binds to ssDNA exposed during replication stress, with and without 40 J UV treatment. Quantification of PCNA-Ub levels normalized to unmodified PCNA, relative to the first lane wild type sample is indicated. Source data for panels b, e, f, g, h, i, and j, including relevant exact p-values, are provided in the Source Data file.

Fig 3: MCM10 heterozygous RPE-1 cell lines have telomere maintenance defects.a Average ß-gal activity in wild type (blue) and clonal MCM10+/- cell lines (red). For each cell line n = 6 replicates. Individual data points are indicated (gray circles). Error bars indicate SD and significance was calculated using an unpaired, two-tailed student’s t-test with *<0.05; **<0.01, ***<0.001. b TRF analysis in wild type and MCM10+/- clone #20 cell lines. PDs and location of peak intensity (yellow dots) are indicated. c TRF analysis in wild type and MCM10+/- clone #20 cell lines in the presence of telomerase inhibitor. PDs and location of peak intensity (yellow dots) are indicated. d TRF analysis in wild type and MCM10 mutant cell lines carrying NK-associated patient variants. Yellow dots indicate the location of peak intensity. Source data for panels a–d, including relevant exact p-values, are provided in the Source Data file.

Fig 4: MCM10 heterozygous RPE-1 cell lines have reduced MCM10 expression, impaired proliferation, and fewer active replication forks.a Western blot for MCM10 with quantification normalized to tubulin relative to wild type is indicated. b Average proliferation rate in MCM10+/- cells normalized to wild type. For each cell line n = 6 replicates. Average values for biological replicates are indicated (gray circles). c Average percentage of early apoptotic, late apoptotic, or dead cells. Wild type (blue) and clonal MCM10+/- cell lines (red) are shown. For each cell line n = 4. Individual data points are indicated (gray circles). d Cell cycle distribution of wild type and MCM10+/- cells. Percentage of each population in G1- (green), S- (purple), and G2/M-phase (gray) is shown. e Average cell cycle distribution of wild type and MCM10+/- cells from three biological replicates. Percentage of each population in G1- (green), S- (purple), and G2/M-phase (gray). Individual data points are indicated (gray circles). f Flow cytometry plots for wild type and MCM10+/- cells. G1-phase/MCM positive cells (blue), S-phase/MCM positive cells (orange) and G1- or G2/M-phase/MCM negative cells (gray) are indicated. g Comparison of origin licensing (left) and average G1 loaded MCM2 in biological replicates (n = 3) of wild type (gray) and MCM10+/- cells (blue). h Comparison of S-phase DNA synthesis (left) and average EdU intensity in biological replicates (n = 3) of wild type (gray) and MCM10+/- cell lines (red). i Chromatin-associated PCNA, PCNA-Ub MCM10, and Lamin B1. Quantification of PCNA-Ub or MCM10 levels normalized to Lamin B1, relative to the first lane is indicated. j Average G1 loaded MCM2 in biological replicates (n = 3) of wild type HCT116 (gray) and RPE-1 (blue) cells. k Average EdU intensity in biological replicates (n = 3) of wild type HCT116 (red) and RPE-1 (gray) cells. Error bars in b, c, e, g, h, and j, k indicate SD and significance was calculated using an unpaired, two-tailed student’s t-test with *<0.05; **<0.01, ***<0.001. Source data for panels a, b, c, e, g, h, i, j, and k, including relevant exact p-values, are provided in the Source Data file.

Fig 5: Telomeric replication stress is increased in ST MCM10+/- HCT116 cells.a Telomere combing images with telomeric DNA (green), nascent DNA (red), and merged images with examples of unreplicated telomeres (left), partially replicated (middle), and completely replicated telomeres (right). Telomeric and sub-telomeric regions are indicated. Scale bars are 3 µm. b Average percentage of unreplicated telomeres in wild type (blue) and MCM10+/- cell lines (red) ST cell lines, n = total number of telomeres quantified. Average values for each replicate are indicated (gray circles). c Average percentage of completely replicated (yellow) versus partially/stalled telomeres (green) in ST cell lines, n = total number of replicated telomeres quantified. Average values for each replicate are indicated (gray circles). Error bars in b and c indicate SD and significance was calculated using an unpaired, two-tailed student’s t-test with *<0.05; **<0.01, ***<0.001. d (Left) Cartoon of t-complex DNA as formerly depicted37. (Middle) Diagram of double-stranded telomere restriction fragment (ds-TRF), telomere circle (t-circle) and telomere complex (t-complex) DNA species from 2D gel electrophoresis. (Right) Comparison of 2D gels from ST cell lines. e Comparison of DNA species from 2D gel electrophoresis in HCT116 wild-type ST cells with and without 4-day HU treatment. f Comparison of DNA species from 2D gels in HCT116 wild type and MCM10+/- ST cell lines with and without S1 nuclease digestion. g (Left) Image of t-SCE staining in ST cell lines. Examples of a t-SCE event and chromosomes without t-SCE are highlighted. (Right) Percentage t-SCE per chromosome ends. Bars represent average percentage t-SCE per chromosome ends; where n = the number of metaphases analyzed. Significance was calculated using an unpaired, two-tailed student’s t-test but were not statistically significant (n.s.). Scale bar is 10 µm. Source data for panels b, c, d, e, f, and g, including relevant exact p-values, are provided in the Source Data file.