Posttranslational modifications (PTMs) of proteins by covalent
attachment of functional groups (like phosphorylation, acetylation,
methylation, glycosylation, etc.) are of key importance for the
cell as they regulate various aspects of protein behavior after its
synthesis, e.g., dictate protein interaction properties, change
catalytic activity of enzymes, induce conformational changes, guide
subcellular localization and determine protein stability. A special
class of protein PTMs is the conjugation of small proteins of the
ubiquitin family to typically acceptor lysine residues of the
substrates. The reversible nature of this PTM and the presence of
dedicated domains that specifically recognize modified substrates
make this type of protein modification instrumental for the
regulation of numerous biological pathways. For ubiquitylation,
strong substrate selectivity due to the presence of highly
diversified conjugation machinery is characteristic and well
studied, especially in case of ubiquitin’s proteolytic role. On the
contrary, much less is known about the principles of substrate
specificity and mechanisms of PTM action in the ubiquitin-like
protein SUMO modification system. Despite the fact that SUMOylation
specifically targets hundreds of substrates and major conjugation
steps are identical with ubiquitin system, strikingly only a
handful of enzymes operate in the SUMO pathway, suggesting that
other principles of substrate selectivity must apply and perhaps
distinct mechanisms of PTM action exist in the SUMO pathway.
Moreover, the recognition of SUMO modification is surprisingly
simple and relies mainly on a short hydrophobic sequence known as
SUMO-interacting motif (SIM), in striking contrast to the ubiquitin
system, where numerous ubiquitin-binding domains exist with
different interaction specificities. All these, together with the
observations that SUMO conjugation machinery seems rather
promiscuous in vitro, that typically only a small fraction of a
protein is being SUMOylated at a given time, and that specific
SUMOylation-defective mutants often exhibit no obvious phenotypes,
whereas SUMO pathway mutants do, emphasize the question of
substrate specificity in the SUMO system and suggest other
principles of SUMO action on its substrates. Here, we address the
question of SUMOylation specificity and function using DNA
double-strand break (DSB) repair pathway via homologous
recombination (HR) as a case study because of its strong ties to
the SUMO system. First, using SILAC-based proteomic approach we
show that proteins acting in the same DNA repair pathway become
collectively SUMOylated upon a specific stimulus (HR factors – upon
DSB induction; nucleotide excision repair factors – upon exposure
to UV light), suggesting that SUMO machinery often targets protein
groups within the same pathway. Then, focusing on the DSB repair we
find that DNA-bound SUMO ligase Siz2 catalyzes collective multisite
SUMOylation of a whole set of HR factors. Repair proteins are
loaded onto resected single-stranded DNA (ssDNA) in the vicinity of
the ligase, thus making exposure of ssDNA a precise trigger for
modification. Protein group SUMOylation fosters physical
interactions between the HR proteins engaged in DNA repair, because
not only that they become collectively modified at multiple
SUMO-acceptor sites, but they also possess multiple SIMs, which
promote SUMO-SIM mediated complex formation. Only wholesale
elimination of SUMOylation of the core HR proteins significantly
affects the HR pathway by slowing down DNA repair, suggesting that
SUMO acts synergistically on several proteins. Thus, we show that
SUMOylation collectively targets functionally engaged protein group
rather than individual proteins, whereas localization of
modification enzymes and specific triggers ensure substrate
specificity.
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