PIAS3 promotes homology-directed repair and distal non-homologous end joining

Abstract

A DNA double-strand break (DSB) is the most severe form of DNA damage and is mainly repaired through homologous recombination (HR), which has a high fidelity, or non-homologous end joining (NHEJ), which is prone to errors. Defects in the DNA damage response lead to genomic instability and ultimately the predisposition of organs to cancer. Protein inhibitor of activated STAT-1 (PIAS1), which is a potential small ubiquitin-related modifier (SUMO) ligase, has been reported to be involved in DSB repair. The present study identified that another member of the PIAS family, PIAS3, is also an enhancer for HR- and NHEJ-mediated DSB repair. Furthermore, the overexpression of PIAS3 was demonstrated to increase the resistance of HeLa cells to ionizing radiation (IR), indicating a significant role for PIAS3 in the DNA damage response (DDR) pathway.

Keywords: PIAS; double-strand break repair; homologous recombination; non-homologous end joining.

Figure 1

Establishment of the EJ5-GFP and DR-GFP systems. (A) DR-GFP is shown along with the HDR product that uses iGFP as the template for nascent DNA synthesis, which results in the restoration of a GFP expression cassette. (B) EJ5-GFP is shown along with products of EJ between the distal DSB ends (distal-EJ) that restores the GFP expression cassette. DR-GFP, direct repeatgreen fluoresecent protein; HDR, homology-directed repair; NHEJ, non-homologous end joining; DSB, double-strand break; iGFP, intense GFP.

Figure 2

Testing the accuracy of EJ5-GFP and direct repeat (DR)-GFP systems using classical factors. (A) ATM specifically inhibits distal-NHEJ DSB repair. The GFP⁺ cell percentages were assayed using FACScan in the control and ATM-knockdown cells. ^*P<0.01 vs. control cells. Western blot analysis of ATM expression in human 293T cells is shown in the lower panel. (B) Two individual cell lines were transfected with an expression vector for I-SceI, along with a complementation vector for BRCA1 or the empty expression vector (EV). Repair is measured as the percentage of GFP⁺ cells, which is normalized to the EV samples transfected in parallel. ^*P<0.001 vs. EV. Representative western blots of BRCA1 expression in human 293T cells is shown in the right panel with BRCA1 carrying HA-HRP. ATM, ataxia telangiectasia mutated gene; NHEJ, non-homologous end joining; DSB, double-strand break; GFP, green fluorescent protein; HRP, horseradish peroxidase.

Figure 3

PIAS3 promotes HDR and distal NHEJ. The human 293T cells were transfected with an expression vector for I-SceI, along with a complementation vector for PIAS1, PIAS3, P1AS1 plus PIAS3 or the empty expression vector (EV). Repair is measured as the percentage of GFP⁺ cells. (^*P<0.001, statistical differences between EV and PIAS1, PIAS3 or P1AS1 plus PIAS3 treatments). Representative western blots of PIAS1 and PIAS3 expression in human 293T cells are shown in the lower panel, with PIAS1 and PIAS3 carrying Myc-HRP. PIAS, protein inhibitor of activated STAT; HDR, homology-directed repair; NHEJ, non-homolous end joining; GFP, green fluorescent protein; HRP, horseradish peroxidase.

Figure 4

Expression of PIAS3 in HeLa cells increases the cell resistance to IR. The surviving fraction of the empty vector Hela cells and the Hela cells overexpressing PIAS3 following exposure to various doses of irradiation is shown. Each point represents the mean surviving fraction. Error bars indicate the standard deviation of two independent biological samples of one experiment. PIAS, protein inhibitor of activated STAT; IR, ionizing radiation; HRP, horseradish peroxidase; EV, empty expression vector.