DOI: 10.5061/dryad.8gtht771b
title: Endogenous p21 levels protect genomic stability by suppressing both excess and restrained nascent DNA synthesis

description: The rate of DNA synthesis is crucial for full DNA duplication. We report a
 key role of p21 in controlling this rate. During normal replication, p21
 promotes nascent DNA synthesis alongside the Pol iota ()/p53 complex. When
 p21 is downregulated but detectable, nascent DNA tracks are longer,
 discontinuous, and rely on PrimPol. With the complete elimination of p21,
 nascent DNA tracks become shorter, continuous, and depend on Pol kappa ().
 Endogenous p21 levels are critical for genomic stability, as both PrimPol
 and Pol-mediated synthesis can induce chromosomal instability. The
 residual expression of p21 in p53 null cells influences the involvement of
 PrimPol or Pol in nascent DNA synthesis and subsequent chromosomal
 instability. Our results demonstrate that endogenous levels of p21 in
 cycling cells, insufficient for cyclin-dependent kinase inhibition,
 prevent genomic instability through PCNA binding, limiting PrimPol and
 Pol's role in nascent DNA synthesis.
description: # Endogenous p21 levels protect genomic stability by suppressing both
 excess and restrained nascent DNA synthesis Dataset DOI:
 [10.5061/dryad.8gtht771b](10.5061/dryad.8gtht771b) The following dataset
 contains numerical data and unprocessed images used during the processing
 of experiments described in an article entitled “Endogenous p21 levels
 protect genomic stability by suppressing both excess and restrained
 nascent DNA synthesis,” which is currently published in an open-access
 source.  The numerical values correspond to quantification of the
 following assays: DNA fiber spreading, quantitative real-time PCR,
 micronucleus assay, anaphase aberration assay, immunostaining and
 fluorescence detection, and DNA polymerase foci detection. They are
 consolidated into a single Excel file with multiple sheets. Each sheet
 corresponds to the data used for a specific experiment as indicated by its
 name (inspired in the processed figure reported in the article). The
 section titled "Description of the data and file structure"
 provides a detailed explanation of the experimental context for each Excel
 sheet. Grey cells: variables as described below. Blue cells: data sets
 used to generate the analysis associated with the title of each sheet.
 This means blue cells indicate the samples used to generate the indicated
 figure. **Description of the data and file structure** The files and their
 respective descriptions are listed below. ## **Files and variables** ####
 **File: Source_data_Excel_file.xlsx** **Description:** The Excel file
 called "Source data Excel file" is a consolidated file
 containing raw data corresponding to quantification of assays as
 described: DNA fiber spreading, quantitative real-time PCR, micronucleus
 assay, anaphase aberration assay, immunostaining and fluorescence
 detection, and DNA polymerase foci detection.
 (Source_data_Excel_file.xlsx). To properly analyze the Excel file, it is
 essential to keep in mind that: 1\)      The Excel cells highlighted in
 light blue in each sheet of the Excel file indicate which part of the data
 from these raw panels was used to generate figures for the above-mentioned
 manuscript. 2\)      All data are organized in Excel sheets named
 according to the figure numbers they correspond to (e.g., Fig. 1A).
 3\)      Each spreadsheet contains the data used to generate the final
 analysis of the described experiment. For each variable, the corresponding
 experiment (1, 2, or 3) is specified where applicable. 4\)      The Excel
 file with raw data does not contain merged cells, images, frozen panes,
 comments, hyperlinks, formulas, filters, or other elements that could
 affect the analysis. The numerical values are associated with the
 following assays, as described: DNA fiber spreading: DNA track length
 (µm). Quantitative real-time PCR: CT values (qPCR cycles) used to
 calculate mRNA expression levels. Micronucleus assay: Number of
 micronuclei relative to the total number of nuclei counted. Anaphase
 aberration assay: Number of anaphase aberrations (bridge or
 lagging/acentric) relative to the total number of anaphases analyzed.
 Immunostaining and fluorescence detection: Signal intensity expressed in
 Arbitrary Units or as a percentage of total nuclei quantified. DNA
 polymerase foci detection: Number of nuclei containing polymerase foci (we
 quantified V5-PrimPol or GFP-Pol k foci) relative to the total number of
 nuclei analyzed. The sheets corresponding to quantifications for the DNA
 fiber spreading assay have the following labels: Fig. 1A, Fig. 1B, Fig.
 1D, Fig. 2B, Fig. 3B, Fig. 5C, Fig. 5D, Fig. 5E, Fig. 5F, Fig. 6D, Fig.
 7C, Fig. 7F, Fig. S1B, Fig. S3A, Fig. S3B, Fig. S4A, Fig. S4B, Fig. S4C,
 Fig. S4D, Fig. S5B, Fig. S5C, Fig. S5E, and Fig. S5G. The sheets
 corresponding to quantitative real-time PCR data have the following
 labels: Fig. 2A, Fig. 5B, Fig. 7B, Fig. S5A, Fig. S5D, Fig. S5F, Fig. S9B,
 and Fig. S11A. The sheets corresponding to micronucleus assay data have
 the following labels: Fig. 4A, Fig. 7D, Fig. 7E, Fig. 7G, Fig. 7H, Fig.
 S8A, Fig. S8D, and Fig. S10A. The Sheet corresponding to the anaphase
 aberration assay has the following label: Fig. 4B. The sheets
 corresponding to immunostaining and fluorescence detection have the
 following labels, which include information on the target protein for each
 immunofluorescence assay in parentheses: Fig. 2H (RPA-foci per nuclei),
 Fig. 3C (BrdU intensity), Fig. 3D (BrdU positive cells), Fig. 3E (gH2AX
 intensity), Fig. 4C (gH2AX intensity), Fig. 4D (ultra-fine bridges by PICH
 protein positive), Fig. 4E (53BP1 nuclear bodies by 53BP1 focis in
 nuclei), Fig. S8B (gH2AX intensity), Fig. S8C (53BP1 nuclear bodies by
 53BP1 focis in nuclei), Fig. S8E (gH2AX intensity), Fig. S10B (gH2AX
 intensity), Fig. S10D (gH2AX intensity), and Fig. S10E (gH2AX intensity).
 The sheets corresponding to DNA polymerase foci detection have the
 following labels and include the specific DNA polymerase for which the
 number of foci per nucleus is quantified, indicated in parentheses: Fig.
 2E (V5-PrimPol foci), Fig. 2F (V5-PrimPol foci), Fig. 2G (V5-PrimPol
 foci), Fig. 3A (V5-PrimPol foci), Fig. 6A (V5-PrimPol foci), Fig. 6C
 (V5-PrimPol foci), Fig. S6B (GFP-Pol k), Fig. S6C (GFP-Pol k), Fig. S6D
 (V5-PrimPol foci), Fig. S6E (GFP-Pol k), and Fig. S11B (V5-PrimPol foci).
 **Variables (highlighted in grey boxes)** The variables contained in the
 aforementioned Excel file are listed below. * siLuc corresponds to the
 control sample with the transduction of siRNA (100nM) against luciferase.
 * sip21 1 low corresponds to the sample with the transduction of siRNA
 (10nM) against p21.  * sip21 1 high corresponds to the sample with the
 transduction of siRNA (100nM) against p21.  * sip21 2 corresponds to the
 sample with the transduction of other siRNA (Ambion, s416) (100nM) against
 p21.  * Olaparib corresponds to the treatment for 24 hs with the drug
 olaparib 10 uM. * siPol eta corresponds to the sample with the
 transduction of siRNA (100nM) against pol eta.  * sip53 corresponds to the
 sample with the transduction of siRNA (100nM) against to p53.  * siIota
 corresponds to the sample with the transduction of siRNA (100nM) against
 pol Iota.  * siPrimPol corresponds to the sample with the transduction of
 siRNA (100nM) against PrimPol.  * siPol k corresponds to the sample with
 the transduction of siRNA (100nM) against Pol k.  * EV corresponds to the
 control sample with the transduction of the empty vector plasmid (Plasmid
 #17482, Addgene) * p21 corresponds to the sample with the transduction of
 the expression vector CS2-p21. * p21PipMut corresponds to the sample with
 the transduction of expression vector CS2-p21 with the M147A, D149A and
 F150A mutations which were shown to disrupt the PCNA/p21 interaction have
 three mutations in its CDK binding domain (W49R, F51S and D52A) which
 disrupt the CDK2/p21 interaction. * p21KO or p21 -/- corresponds to the
 sample of a cell with a p21 knock out. * p53KO or p53 -/- corresponds to
 the sample of a cell with a p53 knock out. * Pol I KO corresponds to the
 sample of a cell with an ADN Pol Iota knock out.  #### **File:
 Source_Western_blot_file_Calzetta_Mansilla_et_al.pdf**
 **Description:** The PDF file called "Source Western blot file
 Calzetta Mansilla et al" contains all raw data corresponding to
 Western Blot images
 (Source_Western_blot_file_Calzetta_Mansilla_et_al.pdf). This file contains
 raw Western blot images, with the specific regions of interest indicated
 by red boxes. To the left of each blot, the protein detected in each
 experiment is specified. In some cases, different exposure times of the
 membrane are also indicated.  #### **File:
 Source_Representative_Images_file_Calzetta_Mansilla_et_al.pdf**
 **Description:** The PDF file called "Source Representative Images
 file Calzetta Mansilla et al" contains raw data corresponding to
 Representative Images related to the Excel file
 (Source_Representative_Images_file_Calzetta_Mansilla_et_al.pdf). This file
 contains the raw images, with specific regions selected for presentation
 indicated by red boxes. ## **Code/software** N/A ## **Access information**
 Other publicly accessible locations of the data: * N/A Data was derived
 from the following sources: * N/A