Small ubiquitin-like modifier (SUMO) isoforms and conjugation-independent function in DNA double-strand break repair pathways

Abstract

Small ubiquitin-like modifier (SUMO) proteins act in DNA double-strand break (DSB) repair, but the pathway specificity of the three major isoforms has not been defined. In experiments in which we depleted the endogenous SUMO protein by RNAi, we found that SUMO1 functioned in all subpathways of either homologous recombination (HR) or non-homologous end joining (NHEJ), whereas SUMO2/3 was required for the major NHEJ pathway, called conservative NHEJ, but dispensable in other DSB repair pathways. To our surprise, we found that depletion of UBC9, the unique SUMO E2 enzyme, had no effect in HR or alternative NHEJ (Alt-NHEJ) but was required for conservative NHEJ. Consistent with this result, both non-conjugatable mutant and wild-type SUMO1 proteins functioned similarly in HR and Alt-NHEJ. These results detail the functional roles of specific SUMO isoforms in DSB repair in mammalian cells and reveal that SUMO1 functions in HR or Alt-NHEJ as a free protein and not as a protein conjugate.

Keywords: DNA Damage Response; Double-strand Break Repair; Homologous Recombination; Non-homologous End Joining; SUMO-interacting Motif (SIM); Small Ubiquitin-like Modifier (SUMO); Sumoylation; UBC9.

FIGURE 1.

SUMO isoforms function differently in DSB repair pathways. A–C, the recombination substrates are diagrammed on the right with details described previously (30–32). iGFP indicates inactive GFP gene. HeLa-derived cell lines for HDR (A), SSA (B), and Alt-NHEJ (C) were subjected to two rounds of siRNA transfection, as indicated, followed by transfection of the I-SceI expression plasmid to induce DSB. After 3 days, the percentages of GFP-positive cells were determined by flow cytometry. In each experiment, the percentage of GFP-positive cells from control siRNA transfections was set equal to 1, and the fraction of GFP-positive cells was determined relative to the control siRNA (Con) to measure HDR, SSA, and Alt-NHEJ, respectively. Results (mean ± S.E.) are from three independent experimental replicates. NT indicates no transfection of the I-SceI-expressing plasmid. D, the C-NHEJ repair substrate in the genome of 293 cells is diagrammed on the right as described previously (33). C-NHEJ assay was done by transfecting cells with the indicated siRNAs as in panel A. After 3 days, the repair efficiency was measured by quantitative real-time PCR on extracted genomic DNA, represented by the percentage on the y axis. In each experiment, the yield of repaired DNA was normalized relative to the value of the result from the control siRNA transfection. E, immunoblots show the depletion of indicated protein by RNAi interference in HeLa cells. Upon siSUMO2/3 transfection, the bottom band (∼15 kDa) of the doublet was depleted. GAPDH and β-actin were used as loading controls. The positions of the molecular mass markers in kDa are indicated at the left. F, results (mean ± S.E.) from each functional DSB repair assay were summarized for the indicated siRNA transfection.

FIGURE 2.

SUMO isoform depletion has no effect on cell cycle progression and DSB repair protein stability. A, endogenous SUMO1, SUMO2/3, or UBC9 was depleted by two rounds of siRNA transfection in HeLa cells. Cell cycle analysis by flow cytometry was carried out at 48, 72, and 96 h after the second transfection. DNA content of the HeLa cells, as determined by staining with propidium iodide, was measured by FACS analysis. B, depletion of SUMO isoforms or UBC9 by two rounds of siRNA transfection was done in HeLa cells as in A. Con, control; S1, SUMO1; S2/3, SUMO2/3. Analysis of immunoblots was applied to measure protein abundance using specific antibody against the DSB repair protein. RNA helicase A (RHA) was used as a loading control, and the positions of the molecular mass markers in kDa are indicated at the left.

FIGURE 3.

Non-conjugated SUMO1 stimulates HR and Alt-NHEJ. A–D, the appropriate cell line was transfected with siSUMO1-3′ targeting the 3′-UTR of the SUMO1 mRNA plus a wild-type SUMO1 or SUMO1-ΔGG expression plasmid or an empty vector and assayed in DSB repair as in Fig. 1. The fraction of GFP-positive HeLa cells was determined by flow cytometry as a measure of repair efficiency of HDR, SSA, and Alt-NHEJ, respectively (A–C), or genomic DNA was extracted from 293 cells for quantitative real-time PCR analysis (D). NT indicates no transfection of the I-SceI-expressing plasmid. Results are mean ± S.E. E, whole cell lysates were extracted from HeLa cells 3 days after the second transfection and subjected to immunoblot analysis. SUMO1 specific antibody detected both endogenous SUMO1 and expressed FLAG-SUMO1 protein. FLAG specific antibody detected FLAG-SUMO1-conjugated protein. The asterisk indicates a nonspecific band. The β-actin protein was a loading control. The positions of the molecular mass markers in kDa are indicated at the left. Vec, vector; Con, control siRNA.

FIGURE 4.

Model for SUMO proteins function in DSB repair. Following DSB, end resection is required for HR or Alt-NHEJ repair; otherwise, DNA ends are protected from minimal processing and C-NHEJ is employed to repair the damage. A, in the HDR pathway, SUMO1 (S1) stimulates the repair process by a non-covalent binding to SIM of RAD51 or other repair proteins. We model that the SUMO1-SIM interaction regulates RAD51 accretion on resected DSB ends. B and C, when SSA or Alt-NHEJ is utilized in response to a DSB, SUMO1 interacts non-covalently with an unknown repair factor (X or Y) via the SIM of the protein at sites of DSB to promote the subsequent repair events. D, in C-NHEJ, in which DNA ends are protected, SUMO1 and SUMO2/3 (S2/3) conjugation to an unknown target repair mediator (Z) is essential for efficient repair.