Small Ubiquitin-like Modifier (SUMO) Isoforms and Conjugation-independent Function in DNA Double-strand Break Repair Pathways

Yiheng Hu,Jeffrey D Parvin

doi:10.1074/jbc.c114.582122

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.

Highlights

The Small ubiquitin-like modifier (SUMO) system is involved in doublestrand break (DSB) repair
We used HeLa- or 293-derived cell lines with the specific recombination substrate DNA integrated in the genome to test the specificity of SUMO isoforms in each DSB repair pathway (Fig. 1, A–D, right). siRNAdependent depletion of each isoform in the appropriate cell line probes the two homologous recombination (HR) pathways, homology-directed repair (HDR) and single-strand annealing (SSA), and the two non-homologous end joining (NHEJ) pathways, Alt-NHEJ and conservative NHEJ (C-NHEJ) (30 –33)
Depletion of SUMO1 (Fig. 1E) reduced repair in all four subpathways tested to about 62% HDR, 31% SSA, 41% Alt-NHEJ, and 39% C-NHEJ, respectively, relative to the control siRNA (Fig. 1, A–D), suggesting that the SUMO1 isoform is stimulatory in all mechanisms of DSB repair

Summary

Background

The SUMO system is involved in doublestrand break (DSB) repair. Results: SUMO2/3 is required for the major NHEJ pathway; SUMO1 stimulates all DSB repair pathways, and a non-conjugatable form of SUMO1 stimulates DSB repair pathways involving DNA end resection. 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. 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 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. We conclude that C-NHEJ is SUMOylation-dependent, the HR and Alt-NHEJ pathways are stimulated by non-covalent SUMO1 interactions

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION