ATPase-Dependent Control of the Mms21 SUMO Ligase during DNA Repair

Abstract

Modification of proteins by SUMO is essential for the maintenance of genome integrity. During DNA replication, the Mms21-branch of the SUMO pathway counteracts recombination intermediates at damaged replication forks, thus facilitating sister chromatid disjunction. The Mms21 SUMO ligase docks to the arm region of the Smc5 protein in the Smc5/6 complex; together, they cooperate during recombinational DNA repair. Yet how the activity of the SUMO ligase is controlled remains unknown. Here we show that the SUMO ligase and the chromosome disjunction functions of Mms21 depend on its docking to an intact and active Smc5/6 complex, indicating that the Smc5/6-Mms21 complex operates as a large SUMO ligase in vivo. In spite of the physical distance separating the E3 and the nucleotide-binding domains in Smc5/6, Mms21-dependent sumoylation requires binding of ATP to Smc5, a step that is part of the ligase mechanism that assists Ubc9 function. The communication is enabled by the presence of a conserved disruption in the coiled coil domain of Smc5, pointing to potential conformational changes for SUMO ligase activation. In accordance, scanning force microscopy of the Smc5-Mms21 heterodimer shows that the molecule is physically remodeled in an ATP-dependent manner. Our results demonstrate that the ATP-binding activity of the Smc5/6 complex is coordinated with its SUMO ligase, through the coiled coil domain of Smc5 and the physical remodeling of the molecule, to promote sumoylation and chromosome disjunction during DNA repair.

Sgs1's roles in DNA end resection, HJ dissolution, and crossover suppression require a two-step SUMO regulation dependent on Smc5/6

“…Upon DNA damage requiring STR function at different steps of HR repair, including DSB end resection, dHJ dissolution, and fork rescue, we propose that Smc5/6 is able to recognize suitable DNA substrates for Sgs1. Binding of Smc5/6 to DNA substrate structures ("Recognition of DNA substrate by Smc5/6") leads to ATP-dependent remodeling of Smc5/6 and activation of the SUMO E3 ligase Mms21, as demonstrated earlier (Bermudez-Lopez et al 2015). Mms21 then SUMOylates several subunits of Smc5/6 ("Auto-SUMOylation of Smc5/6").…”

“…Pull-down analyses were performed essentially as described (Bermudez-Lopez et al 2015). Cells were denatured during harvesting and prior to snap freezing by sequential resuspension of yeast cells in 12% TCA and 1 M Tris-HCl (pH 8.0).…”

“…Coimmunoprecipitation analyses were performed as previously described in Bermudez-Lopez et al (2015). Cells were mechanically broken in 50 mM HEPES, 150 mM KCl, 1.5 mM MgCl 2 , 0.5 mM DTT, and 0.5% Triton X-100 (pH 7.5) supplemented with Complete protease inhibitor cocktail tablets (from Roche).…”

PolySUMOylation by Siz2 and Mms21 triggers relocation of DNA breaks to nuclear pores through the Slx5/Slx8 STUbL

“…While this may be partly through a loss of SUMO recognition, the L247A allele appeared to be a useful tool to perturb the Slx5-SMC5/6 complex interaction without interfering with SMC5/6 complex integrity or its SUMOylation activity. In contrast, the temperature-sensitive smc6-9 allele both disrupts the SMC5/6 complex (Torres-Rosell et al 2005;De Piccoli et al 2006;Sollier et al 2009) and compromises its E3 ligase function (Bermudez-Lopez et al 2015). A summary of the relevant interactions disrupted by Nse5 L247A is shown in Figure 5A.…”

DNA activates the Nse2/Mms21 SUMO E3 ligase in the Smc5/6 complex

“…For example, an N‐terminal SAP domain localize Siz2 on DNA, where it promotes sumoylation of homologous recombination factors (Psakhye & Jentsch, ); additionally, Siz2 binds ssDNA through interaction with the ssDNA binding replication protein A (RPA; Chung & Zhao, ). The Nse2 SUMO ligase, a subunit of the Smc5/6 complex, has also been shown to play key roles in the maintenance of chromosome integrity (Zhao & Blobel, ; Ampatzidou et al , ; Branzei et al , ; Potts et al , ; Pebernard et al , ; Behlke‐Steinert et al , ; Chavez et al , ; Bermúdez‐López et al , ). As Nse2 lacks DNA binding domains, its DNA repair functions require its stable docking onto the Smc5 protein (Duan et al , ; Bermúdez‐López et al , ), and the subsequent association of the Smc5/6 complex with damaged sites (De Piccoli et al , ; Lindroos et al , ; Tapia‐Alveal & O'Connell, ; Bustard et al , ).…”