Journal: Science Advances

Article Title: Endogenous p21 levels protect genomic stability by suppressing both excess and restrained nascent DNA syntheses

doi: 10.1126/sciadv.adw4618

Figure Lengend Snippet: ( A ) Quantitative real-time PCR of PrimPol and Pol κ normalized to GAPDH in U2OS cells. Error bars represent the SD of three technical replicates from one experiment representative of two independent experiments. P < 0.01. ( B ) IdU track lengths from DNA fibers in U2OS cells. Cells were labeled with CldU and IdU for 10 and 30 min, respectively. Approximately 300 DNA fibers obtained from three independent experiments were analyzed for each condition. The bars on top of the distribution clouds indicate the median. P < 0.001. ( C ) Western blot revealing PrimPol and Pol κ levels in U2OS cells with the indicated siRNAs. Actin was used as a loading control. ( D ) Representative image of U2OS cells transfected with the V5-PrimPol expression plasmid with (positive) or without (negative) V5-PrimPol foci (scale bar, 5 μm). ( E and F ) Percentage (means + SD) of U2OS cells with V5-PrimPol foci. Approximately 750 nuclei per sample were analyzed in three independent experiments. P < 0.01. ( G ) Percentage (means + SD) of U2OS cells with V5- PrimPol or V5-PrimPol RBDm foci. Approximately 600 nuclei per sample were analyzed in two independent experiments. P < 0.01. ( H ) Percentage (means + SD) of U2OS cells with more than four RPA foci. Approximately 300 nuclei per sample were analyzed in two independent experiments. P < 0.01. The right panel shows representative images of U2OS cells with less than four RPA foci (negative) or with more than four RPA foci (positive). Scale bar, 10 μm. ( I ) Model. Partial silencing of p21 causes an increase in PrimPol-dependent nascent DNA synthesis. In contrast, complete silencing of p21 causes an augmentation of Pol κ–mediated DNA replication.

Article Snippet: The following antibodies were used: α-p21 1:1000 (Santa Cruz Biotechnology, sc-6246, RRID: AB_628073), α-p53 1:1:1 (phosphate-buffered saline:purified immunoglobulin from clone DO-1:purified immunoglobulin from clone PAB-1801), α-Pol η 1:1000 (Santa Cruz Biotechnology, sc-5592, RRID: AB_2167006), α-Pol κ 1:1000 (Bethyl Laboratories, A301-977A, RRID: AB_1548020), α-PrimPol 1:1000 (Proteintech 29824-1-AP, RRID: AB_2918349), and α-Actin 1:20000 (Sigma-Aldrich, A2066, RRID: AB_476693).

Techniques: Real-time Polymerase Chain Reaction, Labeling, Western Blot, Control, Transfection, Expressing, Plasmid Preparation, DNA Synthesis

Journal: Science Advances

Article Title: Endogenous p21 levels protect genomic stability by suppressing both excess and restrained nascent DNA syntheses

doi: 10.1126/sciadv.adw4618

Figure Lengend Snippet: ( A ) Western blot revealing p53 and p21 in parental HCT116, HCT116 p53 KO, and HCT116 p21 KO cells. Actin was used as a loading control. ( B ) Quantitative real-time PCR of PrimPol and Pol κ normalized to GAPDH. Error bars represent the SD of three technical replicates within one experiment from two independent experiments. P < 0.01. ( C ) IdU track lengths from DNA fibers in HCT116, HCT116 p53 KO, and HCT116 p21 KO cells. CldU: 10 min; IdU: 30 min. Approximately 300 DNA fibers obtained from three independent experiments were analyzed for each condition. The bars indicate the median. P < 0.001. ( D ) IdU track lengths from DNA fibers in HCT116, HCT116 p53 KO, and HCT116 p21 KO cells. CldU: 10 min; IdU: 30 min. Approximately 200 DNA fibers obtained from two independent experiments were analyzed for each condition. The bars indicate the median. P < 0.001. The lower panel shows a Western blot revealed with a p21 antibody. Actin was used as a loading control. ( E ) IdU track lengths from DNA fibers in U2OS cells. CldU: 10 min; IdU: 30 min. Approximately 150 DNA fibers obtained from two independent experiments were measured for each condition. The bars indicate the median. P < 0.001. The lower panel shows p53 and p21 levels in a Western blot. Actin was used as a loading control. ( F ) IdU track lengths from DNA fibers in U2OS WT (clone 18) and U2OS Pol ι KO (clones 13 and 24) cells. CldU: 10 min; IdU: 30 min. Approximately 200 DNA fibers obtained from two independent experiments were analyzed for each condition. The bars indicate the median. P < 0.001. ( G ) Model. p21 regulates PrimPol through Pol ι. However, p21 regulates Pol κ more strongly, and it can only synthesize DNA when p21 is completely depleted.

Article Snippet: The following antibodies were used: α-p21 1:1000 (Santa Cruz Biotechnology, sc-6246, RRID: AB_628073), α-p53 1:1:1 (phosphate-buffered saline:purified immunoglobulin from clone DO-1:purified immunoglobulin from clone PAB-1801), α-Pol η 1:1000 (Santa Cruz Biotechnology, sc-5592, RRID: AB_2167006), α-Pol κ 1:1000 (Bethyl Laboratories, A301-977A, RRID: AB_1548020), α-PrimPol 1:1000 (Proteintech 29824-1-AP, RRID: AB_2918349), and α-Actin 1:20000 (Sigma-Aldrich, A2066, RRID: AB_476693).

Techniques: Western Blot, Control, Real-time Polymerase Chain Reaction, Clone Assay

Journal: Science Advances

Article Title: Endogenous p21 levels protect genomic stability by suppressing both excess and restrained nascent DNA syntheses

doi: 10.1126/sciadv.adw4618

Figure Lengend Snippet: ( A ) Percentage (means + SD) of U2OS cells with V5-PrimPol foci. Approximately 500 nuclei per sample were analyzed in two independent experiments. P < 0.01. ( B ) Western blot revealing Pol η and p21 in parental HCT116 and HCT116 p21 KO cells. Actin was used as a loading control. ( C ) Percentage (means + SD) of HCT116 and HCT116 p21 KO cells with V5-PrimPol foci. Approximately 500 nuclei per sample were analyzed in two independent experiments. P < 0.01. ( D ) IdU track lengths from DNA fibers in HCT116 and HCT116 p21 KO cells. Cells were labeled with CldU and IdU for 10 and 30 min, respectively. Approximately 200 DNA fibers obtained from two independent experiments were analyzed for each condition. The bars on top of the distribution clouds indicate the median. P < 0.001. ( E ) Model. p21 controls PrimPol through Pol ι. However, at high levels, p21 primarily antagonizes Pol κ. Only when p21 is completely depleted, Pols κ and η prevent PrimPol DNA replication.

Article Snippet: The following antibodies were used: α-p21 1:1000 (Santa Cruz Biotechnology, sc-6246, RRID: AB_628073), α-p53 1:1:1 (phosphate-buffered saline:purified immunoglobulin from clone DO-1:purified immunoglobulin from clone PAB-1801), α-Pol η 1:1000 (Santa Cruz Biotechnology, sc-5592, RRID: AB_2167006), α-Pol κ 1:1000 (Bethyl Laboratories, A301-977A, RRID: AB_1548020), α-PrimPol 1:1000 (Proteintech 29824-1-AP, RRID: AB_2918349), and α-Actin 1:20000 (Sigma-Aldrich, A2066, RRID: AB_476693).

Techniques: Western Blot, Control, Labeling

Journal: Science Advances

Article Title: Endogenous p21 levels protect genomic stability by suppressing both excess and restrained nascent DNA syntheses

doi: 10.1126/sciadv.adw4618

Figure Lengend Snippet: ( A ) Western blot revealing p53 and p21 levels in H1299 and SKOV3 cells. Actin was used as a loading control. sip21#1 (100 nM) was transfected into each cell line to achieve complete p21 down-regulation. ( B ) Quantitative real-time PCR of PrimPol and Pol κ normalized to GAPDH in H1299 and SKOV3 cells. Error bars represent the SD of three technical replicates from one experiment representative of two independent experiments. P < 0.01. ( C ) IdU track lengths from DNA fibers in H1299 cells. Cells were labeled with CldU and IdU for 10 and 30 min, respectively. Approximately 200 DNA fibers obtained from two independent experiments were analyzed for each condition. The bars on top of the distribution clouds indicate the median. P < 0.001. ( D ) Percentage (means + SD) of binucleated H1299 cells with micronuclei. Approximately 300 binucleated cells per sample were analyzed in two independent experiments. P < 0.01. ( E ) Percentage (means + SD) of binucleated H1299 cells with micronuclei. sip21#1 (100 nM) was transfected to achieve complete p21 down-regulation. Approximately 300 binucleated cells per sample were analyzed in two independent experiments. P < 0.01. ( F to H ) The IdU track length from DNA fibers and the percentage of binucleated SKOV3 cells with micronuclei treated with siRNAs were determined as in (C) to (E). P values for (F) to (H) are <0.001, 0.01, and 0.01, respectively.

Article Snippet: The following antibodies were used: α-p21 1:1000 (Santa Cruz Biotechnology, sc-6246, RRID: AB_628073), α-p53 1:1:1 (phosphate-buffered saline:purified immunoglobulin from clone DO-1:purified immunoglobulin from clone PAB-1801), α-Pol η 1:1000 (Santa Cruz Biotechnology, sc-5592, RRID: AB_2167006), α-Pol κ 1:1000 (Bethyl Laboratories, A301-977A, RRID: AB_1548020), α-PrimPol 1:1000 (Proteintech 29824-1-AP, RRID: AB_2918349), and α-Actin 1:20000 (Sigma-Aldrich, A2066, RRID: AB_476693).

Techniques: Western Blot, Control, Transfection, Real-time Polymerase Chain Reaction, Labeling

Journal: Science Advances

Article Title: Endogenous p21 levels protect genomic stability by suppressing both excess and restrained nascent DNA syntheses

doi: 10.1126/sciadv.adw4618

Figure Lengend Snippet: ( A ) Left panel: The endogenous levels of p21 in cells undergoing DNA replication inhibit Pol κ and PrimPol participation in nascent DNA elongation, ensuring a safe rate of DNA replication (green region in the speed meter). Genomic stability is at its highest. Middle panel: Partial depletion of p21 increases the nascent DNA elongation rate because of excess PrimPol participation in DNA replication (dangerously high region in the speed meter). PrimPol-mediated DNA replication does not induce replication stress but induces genomic instability. Right panel: Complete elimination of p21 reduces the nascent DNA elongation rate because of excess participation of Pol κ in DNA replication. Pol κ–mediated DNA replication triggers replication stress and genomic instability. ( B ) The participation of PrimPol in DNA synthesis is subject to strong regulation. p21 prevents PrimPol from participating in nascent DNA synthesis in concert with the Pol ι/p53 DNA replication complex. When p21 is completely down-regulated, Pols κ and η act in concert to prevent PrimPol-dependent DNA synthesis. Only when p21 is partially down-regulated, a window of opportunity for PrimPol is created because the Pol ι/p53 complex is not favored and the participation of Pols κ and η in DNA replication is still prevented by p21.

Article Snippet: The following antibodies were used: α-p21 1:1000 (Santa Cruz Biotechnology, sc-6246, RRID: AB_628073), α-p53 1:1:1 (phosphate-buffered saline:purified immunoglobulin from clone DO-1:purified immunoglobulin from clone PAB-1801), α-Pol η 1:1000 (Santa Cruz Biotechnology, sc-5592, RRID: AB_2167006), α-Pol κ 1:1000 (Bethyl Laboratories, A301-977A, RRID: AB_1548020), α-PrimPol 1:1000 (Proteintech 29824-1-AP, RRID: AB_2918349), and α-Actin 1:20000 (Sigma-Aldrich, A2066, RRID: AB_476693).

Techniques: DNA Synthesis