Abstract
Protein sumoylation, especially when catalyzed by the Mms21 SUMO E3 ligase, plays a major role in suppressing duplication-mediated gross chromosomal rearrangements (dGCRs). How Mms21 targets its substrates in the cell is insufficiently understood. Here, we demonstrate that Esc2, a protein with SUMO-like domains (SLDs), recruits the Ubc9 SUMO conjugating enzyme to specifically facilitate Mms21-dependent sumoylation and suppress dGCRs. The D430R mutation in Esc2 impairs its binding to Ubc9 and causes a synergistic growth defect and accumulation of dGCRs with mutations that delete the Siz1 and Siz2 E3 ligases. By contrast, esc2-D430R does not appreciably affect sensitivity to DNA damage or the dGCRs caused by the catalytically inactive mms21-CH. Moreover, proteome-wide analysis of intracellular sumoylation demonstrates that esc2-D430R specifically down-regulates sumoylation levels of Mms21-preferred targets, including the nucleolar proteins, components of the SMC complexes and the MCM complex that acts as the catalytic core of the replicative DNA helicase. These effects closely resemble those caused by mms21-CH, and are relatively unaffected by deleting Siz1 and Siz2. Thus, by recruiting Ubc9, Esc2 facilitates Mms21-dependent sumoylation to suppress the accumulation of dGCRs independent of Siz1 and Siz2.
Highlights
Segmental duplications are “at-risk” DNA sequences that can cause genome rearrangements through non-allelic recombination pathways
The esc2Δ mms21-CH double mutant grows slower than either single mutant and is hypersensitive to 50 mM HU, while the esc2Δ mutant is not appreciably sensitive at this dosage of HU. This indicates that Mms21 plays a more important role in dealing with DNA replication stress compared to Esc2, and that they share a partially overlapping role in maintaining cell growth
Esc2 and Mms21 appear to act in the same pathway to regulate intracellular sumoylation, and this function is best evaluated using cells lacking the Siz1 and Siz2 Small Ubiquitin-like MOdifer (SUMO) E3 ligases in which Mms21 is the sole remaining mitotic E3 ligase [9]
Summary
Segmental duplications are “at-risk” DNA sequences that can cause genome rearrangements through non-allelic recombination pathways. Previous studies demonstrate that specific pathways are involved in preventing the accumulation of duplication-mediated genome rearrangements [1,2,3]. These pathways involve genes that act during DNA replication and repair, and genes that are involved in post-translational protein modifications. Among the latter, we found that modifications by Small Ubiquitin-like MOdifer (SUMO) play a highly specific and significant role in suppressing duplication mediated gross chromosomal rearrangements (dGCRs) [4, 5]. Saccharomyces cerevisiae expresses three mitotic SUMO E3 ligases, Siz, Siz
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