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A SUMO-interacting motif activates budding yeast ubiquitin ligase Rad18 towards SUMO-modified PCNA.

Parker JL, Ulrich HD - Nucleic Acids Res. (2012)

Bottom Line: Although not completely dependent on sumoylation, Rad18's activity towards PCNA is strongly enhanced by the presence of SUMO on the clamp.The stimulation is brought about by a SUMO-interacting motif in Rad18, which also mediates sumoylation of Rad18 itself.Our results imply that sumoylated PCNA is the physiological ubiquitylation target of budding yeast Rad18 and suggest a new mechanism by which the transition from S phase-associated sumoylation to damage-induced ubiquitylation of PCNA is accomplished.

View Article: PubMed Central - PubMed

Affiliation: Clare Hall Laboratories, Cancer Research UK London Research Institute, Blanche Lane, South Mimms, Hertfordshire EN6 3LD, UK.

ABSTRACT
SUMO-targeted ubiquitin ligases (STUbLs) recognize sumoylated proteins as substrates for ubiquitylation and have been implicated in several aspects of DNA repair and the damage response. However, few physiological STUbL substrates have been identified, and the relative importance of SUMO binding versus direct interactions with the substrate remains a matter of debate. We now present evidence that the ubiquitin ligase Rad18 from Saccharomyces cerevisiae, which monoubiquitylates the sliding clamp protein proliferating cell nuclear antigen (PCNA) in response to DNA damage, exhibits the hallmarks of a STUbL. Although not completely dependent on sumoylation, Rad18's activity towards PCNA is strongly enhanced by the presence of SUMO on the clamp. The stimulation is brought about by a SUMO-interacting motif in Rad18, which also mediates sumoylation of Rad18 itself. Our results imply that sumoylated PCNA is the physiological ubiquitylation target of budding yeast Rad18 and suggest a new mechanism by which the transition from S phase-associated sumoylation to damage-induced ubiquitylation of PCNA is accomplished.

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Covalent and non-covalent interactions of Rad18 with Smt3. (A) Rad189myc is sumoylated in vivo in a SIM-dependent manner. Total Smt3 conjugates were isolated by Ni–NTA pull-down from a strain expressing HisSmt3, and sumoylated Rad189myc (marked ‘S’) was detected in the isolated material by anti-myc western blot. Endogenous ubiquitylated Rad18 (‘U’) is also detectable. (B) Rad18 is sumoylated in vitro. Sumoylation assays were set up under standard conditions with the indicated components, and modified Rad18 was detected by western blot. The ‘10×’ indicates a 10-fold higher concentration of Ubc9. The presence of ubiquitylated Rad18 is a consequence of producing the protein in yeast. (C) Rad18 sumoylation is enhanced by the SIM. Time course analysis of in vitro sumoylation was performed with WT Rad18 and the SIM* mutant protein. (D) Rad18 interacts non-covalently with Smt3 in a SIM-dependent manner. GSTPCNA, GSTSmt3 or GSTUbc9 was immobilized on glutathione Sepharose, and retention of HisRad18(1-255), either WT or SIM*, was analysed by anti-His western blot. GST was used as a negative control.
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gks892-F2: Covalent and non-covalent interactions of Rad18 with Smt3. (A) Rad189myc is sumoylated in vivo in a SIM-dependent manner. Total Smt3 conjugates were isolated by Ni–NTA pull-down from a strain expressing HisSmt3, and sumoylated Rad189myc (marked ‘S’) was detected in the isolated material by anti-myc western blot. Endogenous ubiquitylated Rad18 (‘U’) is also detectable. (B) Rad18 is sumoylated in vitro. Sumoylation assays were set up under standard conditions with the indicated components, and modified Rad18 was detected by western blot. The ‘10×’ indicates a 10-fold higher concentration of Ubc9. The presence of ubiquitylated Rad18 is a consequence of producing the protein in yeast. (C) Rad18 sumoylation is enhanced by the SIM. Time course analysis of in vitro sumoylation was performed with WT Rad18 and the SIM* mutant protein. (D) Rad18 interacts non-covalently with Smt3 in a SIM-dependent manner. GSTPCNA, GSTSmt3 or GSTUbc9 was immobilized on glutathione Sepharose, and retention of HisRad18(1-255), either WT or SIM*, was analysed by anti-His western blot. GST was used as a negative control.

Mentions: We therefore expressed a His7-tagged form of Smt3 in a strain bearing a 9myc-tagged allele of RAD18 to isolate total Smt3 conjugates under fully denaturing conditions. High-molecular-weight forms of Rad189myc were detectable in the isolated material, indicating that Rad18 is indeed covalently sumoylated in vivo (Figure 2A). Modification of the mutant Rad18(SIM*) protein was much reduced, although not completely abolished. Similar results were obtained in vitro, where addition of SUMO-specific E1 and E2 (Aos1-Uba2 and Ubc9) promoted sumoylation of purified Rad18 (Figure 2B). The reaction was independent of the presence of DNA, which is known to be required for Rad18’s ubiquitin ligase activity towards PCNA (30). Rad18’s cognate E2, Rad6, was not modified, and the SUMO ligase Siz1 moderately enhanced the reaction (Supplementary Figure S1). Importantly, the SIM* mutant was sumoylated much less efficiently (Figure 2C).Figure 2.


A SUMO-interacting motif activates budding yeast ubiquitin ligase Rad18 towards SUMO-modified PCNA.

Parker JL, Ulrich HD - Nucleic Acids Res. (2012)

Covalent and non-covalent interactions of Rad18 with Smt3. (A) Rad189myc is sumoylated in vivo in a SIM-dependent manner. Total Smt3 conjugates were isolated by Ni–NTA pull-down from a strain expressing HisSmt3, and sumoylated Rad189myc (marked ‘S’) was detected in the isolated material by anti-myc western blot. Endogenous ubiquitylated Rad18 (‘U’) is also detectable. (B) Rad18 is sumoylated in vitro. Sumoylation assays were set up under standard conditions with the indicated components, and modified Rad18 was detected by western blot. The ‘10×’ indicates a 10-fold higher concentration of Ubc9. The presence of ubiquitylated Rad18 is a consequence of producing the protein in yeast. (C) Rad18 sumoylation is enhanced by the SIM. Time course analysis of in vitro sumoylation was performed with WT Rad18 and the SIM* mutant protein. (D) Rad18 interacts non-covalently with Smt3 in a SIM-dependent manner. GSTPCNA, GSTSmt3 or GSTUbc9 was immobilized on glutathione Sepharose, and retention of HisRad18(1-255), either WT or SIM*, was analysed by anti-His western blot. GST was used as a negative control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC3526273&req=5

gks892-F2: Covalent and non-covalent interactions of Rad18 with Smt3. (A) Rad189myc is sumoylated in vivo in a SIM-dependent manner. Total Smt3 conjugates were isolated by Ni–NTA pull-down from a strain expressing HisSmt3, and sumoylated Rad189myc (marked ‘S’) was detected in the isolated material by anti-myc western blot. Endogenous ubiquitylated Rad18 (‘U’) is also detectable. (B) Rad18 is sumoylated in vitro. Sumoylation assays were set up under standard conditions with the indicated components, and modified Rad18 was detected by western blot. The ‘10×’ indicates a 10-fold higher concentration of Ubc9. The presence of ubiquitylated Rad18 is a consequence of producing the protein in yeast. (C) Rad18 sumoylation is enhanced by the SIM. Time course analysis of in vitro sumoylation was performed with WT Rad18 and the SIM* mutant protein. (D) Rad18 interacts non-covalently with Smt3 in a SIM-dependent manner. GSTPCNA, GSTSmt3 or GSTUbc9 was immobilized on glutathione Sepharose, and retention of HisRad18(1-255), either WT or SIM*, was analysed by anti-His western blot. GST was used as a negative control.
Mentions: We therefore expressed a His7-tagged form of Smt3 in a strain bearing a 9myc-tagged allele of RAD18 to isolate total Smt3 conjugates under fully denaturing conditions. High-molecular-weight forms of Rad189myc were detectable in the isolated material, indicating that Rad18 is indeed covalently sumoylated in vivo (Figure 2A). Modification of the mutant Rad18(SIM*) protein was much reduced, although not completely abolished. Similar results were obtained in vitro, where addition of SUMO-specific E1 and E2 (Aos1-Uba2 and Ubc9) promoted sumoylation of purified Rad18 (Figure 2B). The reaction was independent of the presence of DNA, which is known to be required for Rad18’s ubiquitin ligase activity towards PCNA (30). Rad18’s cognate E2, Rad6, was not modified, and the SUMO ligase Siz1 moderately enhanced the reaction (Supplementary Figure S1). Importantly, the SIM* mutant was sumoylated much less efficiently (Figure 2C).Figure 2.

Bottom Line: Although not completely dependent on sumoylation, Rad18's activity towards PCNA is strongly enhanced by the presence of SUMO on the clamp.The stimulation is brought about by a SUMO-interacting motif in Rad18, which also mediates sumoylation of Rad18 itself.Our results imply that sumoylated PCNA is the physiological ubiquitylation target of budding yeast Rad18 and suggest a new mechanism by which the transition from S phase-associated sumoylation to damage-induced ubiquitylation of PCNA is accomplished.

View Article: PubMed Central - PubMed

Affiliation: Clare Hall Laboratories, Cancer Research UK London Research Institute, Blanche Lane, South Mimms, Hertfordshire EN6 3LD, UK.

ABSTRACT
SUMO-targeted ubiquitin ligases (STUbLs) recognize sumoylated proteins as substrates for ubiquitylation and have been implicated in several aspects of DNA repair and the damage response. However, few physiological STUbL substrates have been identified, and the relative importance of SUMO binding versus direct interactions with the substrate remains a matter of debate. We now present evidence that the ubiquitin ligase Rad18 from Saccharomyces cerevisiae, which monoubiquitylates the sliding clamp protein proliferating cell nuclear antigen (PCNA) in response to DNA damage, exhibits the hallmarks of a STUbL. Although not completely dependent on sumoylation, Rad18's activity towards PCNA is strongly enhanced by the presence of SUMO on the clamp. The stimulation is brought about by a SUMO-interacting motif in Rad18, which also mediates sumoylation of Rad18 itself. Our results imply that sumoylated PCNA is the physiological ubiquitylation target of budding yeast Rad18 and suggest a new mechanism by which the transition from S phase-associated sumoylation to damage-induced ubiquitylation of PCNA is accomplished.

Show MeSH
Related in: MedlinePlus