Department of Cellular and Molecular Medicine, University of California School of Medicine, San Diego, La Jolla, California, United States of America.
Moores-UCSD Cancer Center, University of California School of Medicine, San Diego, La Jolla, California, United States of America.
PLoS One. 2021 Feb 18;16(2):e0247132. doi: 10.1371/journal.pone.0247132. eCollection 2021.
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.
蛋白质 SUMO 化修饰，尤其是由 Mms21 SUMO E3 连接酶催化的 SUMO 化修饰，在抑制复制介导的染色体大片段重排（dGCRs）方面起着重要作用。然而，Mms21 如何在细胞中靶向其底物还不够清楚。在这里，我们证明了具有 SUMO 样结构域（SLDs）的蛋白 Esc2 招募 Ubc9 SUMO 连接酶，以特异性促进 Mms21 依赖性 SUMO 化修饰，并抑制 dGCRs 的发生。Esc2 中的 D430R 突变会损害其与 Ubc9 的结合能力，导致与缺失 Siz1 和 Siz2 E3 连接酶的突变体协同生长缺陷和 dGCRs 积累。相比之下，esc2-D430R 不会显著影响对 DNA 损伤的敏感性，也不会显著影响由催化失活的 mms21-CH 引起的 dGCRs。此外，对细胞内 SUMO 化修饰的蛋白质组分析表明，esc2-D430R 特异性地下调了 Mms21 偏好的靶标（包括核仁蛋白、SMC 复合物的组成部分以及作为复制 DNA 解旋酶催化核心的 MCM 复合物）的 SUMO 化修饰水平。这些影响与 mms21-CH 引起的影响非常相似，而且不受 Siz1 和 Siz2 的删除影响。因此，通过招募 Ubc9，Esc2 促进了 Mms21 依赖性 SUMO 化修饰，从而抑制了 dGCRs 的积累，这一过程独立于 Siz1 和 Siz2。
