SUMOylation regulates telomere length homeostasis by targeting Cdc13

Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 07/2011; 18(8):920-6. DOI: 10.1038/nsmb.2100
Source: PubMed


Telomere length homeostasis is an important aspect of telomere biology. Here, we show that SUMOylation limits telomere length and targets multiple telomere proteins in Saccharomyces cerevisiae. A main target is Cdc13, which both positively and negatively regulates telomerase and confers end protection. We demonstrate that Cdc13 SUMOylation restrains telomerase functions by promoting Cdc13 interaction with the telomerase inhibitor Stn1 without affecting end protection. Mutation of the Cdc13 SUMOylation site (cdc13-snm) lengthens telomeres and reduces the Stn1 interaction, whereas Cdc13-SUMO fusion has the opposite effects. cdc13-snm's effect on telomere length is epistatic with stn1, but not with yku70, tel1 or est1 alleles, and is suppressed by Stn1 overexpression. Cdc13 SUMOylation peaks in early-mid S phase, prior to its known Cdk1-mediated phosphorylation, and the two modifications act antagonistically, suggesting that the opposite roles of Cdc13 in telomerase regulation can be separated temporally and regulated by distinct modifications.

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    • "Despite these studies, further studies are needed to understand the function of E1 and E3 sumoylation. Hundreds of proteins are known to be sumoylated in S. cerevisiae[16,17,20212223242526, and the specific sites at which they are modified have been identified in some cases272829303132333435. One approach for identifying sumoylation sites has been the use of a consensus sumoylation motif to predict candidate sumoylation sites that are then evaluated by mutagenesis studies, i.e., the ψKX(D/E) consensus motif, where ψ is a large hydrophobic residue and X is any amino acid[27]. "
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    ABSTRACT: A variety of cellular pathways are regulated by protein modifications with ubiquitin-family proteins. SUMO, the Small Ubiquitin-like MOdifier, is covalently attached to lysine on target proteins via a cascade reaction catalyzed by E1, E2, and E3 enzymes. A major barrier to understanding the diverse regulatory roles of SUMO has been a lack of suitable methods to identify protein sumoylation sites. Here we developed a mass-spectrometry (MS) based approach combining chemical and enzymatic modifications to identify sumoylation sites. We applied this method to analyze the auto-sumoylation of the E1 enzyme in vitro and compared it to the GG-remnant method using Smt3-I96R as a substrate. We further examined the effect of smt3-I96R mutation in vivo and performed a proteome-wide analysis of protein sumoylation sites in Saccharomyces cerevisiae. To validate these findings, we confirmed several sumoylation sites of Aos1 and Uba2 in vivo. Together, these results demonstrate that our chemical and enzymatic method for identifying protein sumoylation sites provides a useful tool and that a combination of methods allows a detailed analysis of protein sumoylation sites.
    Full-text · Article · Dec 2015 · PLoS ONE
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    • "Sumoylated proteins were detected as in Hang et al. (2011). In brief, mid-log phase cells were treated with 0.03% or 0.3% MMS for 2 hr and lysed in denaturing conditions followed by immunoprecipitation. "
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    ABSTRACT: Elucidating the individual and collaborative functions of genome maintenance factors is critical for understanding how genome duplication is achieved. Here, we investigate a conserved scaffold in budding yeast, Rtt107, and its three partners: a SUMO E3 complex, a ubiquitin E3 complex, and Slx4. Biochemical and genetic findings show that Rtt107 interacts separately with these partners and contributes to their individual functions, including a role in replisome sumoylation. We also provide evidence that Rtt107 associates with replisome components, and both itself and its associated E3s are important for replicating regions far from initiation sites. Corroborating these results, replication defects due to Rtt107 loss and genotoxic sensitivities in mutants of Rtt107 and its associated E3s are rescued by increasing replication initiation events through mutating two master repressors of late origins, Mrc1 and Mec1. These findings suggest that Rtt107 functions as a multi-functional platform to support replication progression with its partner E3 enzymes.
    Full-text · Article · Oct 2015 · Molecular cell
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    • "For Figure 1A, UV and MMS doses were chosen based on comparable survival posttreatment as shown in Supplementary Figure S1A–B. Co-immunoprecipitation experiments were performed as described previously (51). "
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    ABSTRACT: The Saccharomyces cerevisiae Rad1-Rad10 complex is a conserved, structure-specific endonuclease important for repairing multiple types of DNA lesions. Upon recruitment to lesion sites, Rad1-Rad10 removes damaged sequences, enabling subsequent gap filling and ligation. Acting at mid-steps of repair, the association and dissociation of Rad1-Rad10 with DNA can influence repair efficiency. We show that genotoxin-enhanced Rad1 sumoylation occurs after the nuclease is recruited to lesion sites. A single lysine outside Rad1's nuclease and Rad10-binding domains is sumoylated in vivo and in vitro. Mutation of this site to arginine abolishes Rad1 sumoylation and impairs Rad1-mediated repair at high doses of DNA damage, but sustains the repair of a single double-stranded break. The timing of Rad1 sumoylation and the phenotype bias toward high lesion loads point to a post-incision role for sumoylation, possibly affecting Rad1 dissociation from DNA. Indeed, biochemical examination shows that sumoylation of Rad1 decreases the complex's affinity for DNA without affecting other protein properties. These findings suggest a model whereby sumoylation of Rad1 promotes its disengagement from DNA after nuclease cleavage, allowing it to efficiently attend to large numbers of DNA lesions.
    Full-text · Article · Apr 2014 · Nucleic Acids Research
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