SUMOylation of proteins is a cyclic process that requires both conjugation and deconjugation of SUMO moieties. Besides modification by a single SUMO, SUMO chains have also been observed, yet the dynamics of SUMO conjugation/deconjugation remain poorly understood. Using a non-deconjugatable form of SUMO we demonstrate the underappreciated existence of SUMO chains in vivo, we highlight the importance of SUMO deconjugation, and we demonstrate the highly dynamic nature of the SUMO system. We show that SUMO-specific proteases (SENPs) play a crucial role in the dynamics of SUMO chains in vivo by constant deconjugation. Preventing deSUMOylation in Schizosaccharomyces pombe results in slow growth and a sensitivity to replication stress, highlighting the biological requirement for deSUMOylation dynamics. Furthermore, we present the mechanism of SUMO chain deconjugation by SENPs, which occurs via a stochastic mechanism, resulting in cleavage anywhere within a chain. Our results offer mechanistic insights into the workings of deSUMOylating proteases and highlight their importance in the homeostasis of (poly)SUMO-modified substrates.Reversible post-translational modification of proteins by ubiquitin-like covalent modifiers is a widely utilized mechanism to alter the fate, binding partners, function, or localization of a given target protein (1). A prime example is the covalent attachment of multiple ubiquitin moieties to a lysine side-chain of a target protein (2), leading to the formation of ubiquitin chains, which have different functions depending on the lysine utilized in the internal ubiquitin linkage. Recently, small ubiquitin-like modifier (SUMO) 2 has also been shown to form chains in vitro (3, 4) and in vivo (5, 6).The SUMO conjugation pathway consists of SUMO activation, transfer, and ligation enzyme machinery analogous to the ubiquitin pathway (7-9). Somewhere upwards of 500 cellular proteins are SUMOylated in vivo (10), yet the extent of observable SUMOylation is only a snapshot due to the presence of SUMO-specific proteases, SENPs. The SUMO cycle begins and ends with specific proteolytic events: the processing of pro-SUMO and the deconjugation of SUMO from the target protein by SENPs (11). In addition to monoSUMOylation, there is now growing evidence that SUMO, like ubiquitin, forms polymeric chains. Thus, the non-covalent interaction of the SUMO conjugating enzyme, Ubc9, with SUMO has presented a possible mechanism for SUMO chain formation (4, 12) and indeed in vitro, all SUMO molecules (Smt3, the Saccharomyces cerevisiae SUMO homologue, and human SUMO1, -2, and -3) have been observed to form SUMO chains, primarily via lysine residues located near their N termini (13). In vitro the SUMOylation system is quite permissive, allowing for multiple . The initial indication that SUMO chains exist in vivo came from transfection experiments yielding multimers of SUMO2 conjugated to HDAC4 (3) and from S. cerevisiae (15). More recently, evidence for in vivo SUMO chains comes from mass-spectrometry experiments using HeLa ...