Principles of protein group SUMO modification substantiated in DNA repair

Psakhye, Ivan (2013): Principles of protein group SUMO modification substantiated in DNA repair. Dissertation, LMU München: Fakultät für Biologie

Abstract

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.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: protein group modification, ubiquitin family proteins, SUMO, homologous recombination, DNA double-strand break repair, nuclear assemblies, substrate specificity
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 29. Oktober 2013
• 1. Bericht­erstatter:in: Jentsch, Stefan