Fig. 1
From: Mutational signatures reveal the role of RAD52 in p53-independent p21-driven genomic instability
Reduction of single nucleotide substitution (SNS) and malfunction of the translesion DNA synthesis and repair (TLS) process upon protracted p21WAF1/Cip1 expression. a Chronic p21WAF1/Cip1 expression, in a p53-deficient environment, leads to the emergence of a subpopulation of p21WAF1/Cip1 aggressive and chemo-resistant (escaped (ESC)) cells, after bypassing an initial senescence-like phase, that carry a lower SNS “load” [9]. SNS identification and filtering were performed with the use of Samtools and VCFtools in non-induced and escaped (Saos2- and Li-Fraumeni-p21WAF1/Cip1 Tet-ON) cells (see also Additional file 1: Figure S1), depicted in accompanying histograms (*p < 0.05 (Saos2 and Li-Fraumeni), OFF vs ESC, Welch’s t-test) (for details see “Methods” section). b TLS pathway function. TLS is a DNA damage tolerance process enabling the DNA replication machinery to replicate over DNA lesions. Upon DNA damage PCNA is mono-ubiquitinated, followed by polymerase switch from normal high-fidelity DNA replication polymerases to TLS ones. TLS polymerase Polη, bound to PCNA, inserts a nucleotide opposite to the lesion and, assisted or not by an additional TLS polymerase like Polκ or Polζ, extends beyond the insertion. Finally, a second polymerase switch takes place by substituting TLS polymerases with high-fidelity ones. c Sustained p21WAF1/Cip1 expression results in decreased mono-ubiquitination of PCNA (mono-Ub). Immunoblots (IBs) in 96-h induced Saos2- and Li-Fraumeni-p21WAF1/Cip1 Tet-ON cells (n = 3 experiments). d Reduced binding of Polη to chromatin in cells with protracted p21WAF1/Cip1 expression. IBs after cell fractionation (described in scheme) depicting lower levels of Polη in chromatin extracts from 96-h induced Saos2- and Li-Fraumeni-p21WAF1/Cip1 Tet-ON cells (n = 3 experiments). e Immunofluorescent confocal microscopy (top panel) showing reduced Polκ loading on regions of damaged chromatin after UV-laser ablation in 96-h induced Saos2-p21WAF1/Cip1 Tet-ON cells transfected with a GFP-Polκ vector. Plots (lower panel) depict recruitment kinetics of Polκ in Saos2- and Li-Fraumeni- p21WAF1/Cip1 Tet-ON cells, respectively (see also Additional files 2, 3, 4 and 5). The average intensity of fluorescence at the site of damage and the total cell fluorescence with respect to time were quantified and plotted. Five cells in each condition of three independent experiments were processed. Time frames for obtaining IFs and recruitment plots are depicted in middle panel. f A specific p21WAF1/Cip1 mutant (p21PCNA; harboring Q144, M147, F150 substitutions to A in its PCNA-interacting-protein (PIP) degron motif) with an abrogated interaction with PCNA [9]. IBs depict mono-ubiquitination of PCNA (mono-Ub) in 96-h induced Saos2- and Li-Fraumeni-p21PCNA Tet-ON cells (n = 3 experiments). g Overexpression of p21WAF1/Cip1 decreases TLS repair efficiency across a site-specific lesion in a gapped plasmid TLS assay (i). Induced Li-Fraumeni-p21WAF1/Cip1 Tet-ON cells were assayed for TLS efficiency (ii) and accuracy of repair (iii) with a gap-lesion vector carrying a site-specific benzo[a]pyrene-guanine (BP-G) adduct (i) (Additional file 6: Table S1) (n = 3 experiments). Actin and lamin B serve as loading control (* p < 0.05, error bars indicate SDs). MQ mapping quality, AF allele frequency, DP sequencing depth, MCM Mini-Chromosome Maintenance protein complex, RPA Replication Protein A, WCE whole cell extract, S2 soluble cytosolic, S3 soluble nuclear, P3 chromatin-nuclear matrix