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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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Relevance of the SIM–SUMO interaction for Rad18 function in vivo. (A) Damage-induced PCNA ubiquitylation is reduced in the SIM* mutant. HisPCNA was isolated by Ni–NTA pull-down under denaturing conditions from extracts of MMS-treated cells, and ubiquitin conjugates were detected by anti-ubiquitin western blot. (B) Mutation of the SIM confers enhanced sensitivity to MMS in a manner dependent on K164 of PCNA, but the phenotype is suppressed by deletion of SIZ1. Deletion mutants of rad18 were complemented with RAD18 (WT or SIM*) for spot assays. An empty vector served as control. (C) Mutation of the SIM confers enhanced UV sensitivity. (D) Damage-induced PCNA ubiquitylation is reduced in a mutant deficient in PCNA sumoylation, pol30(K127R) siz1. A modification time course was analysed as in panel A. (E) Quantification of the blots shown in panel D, relative to the WT signal at 60 min. (F) The pol30(K127R) siz1 mutant displays enhanced UV sensitivity. (G) The PCNA ubiquitylation defects of rad18(SIM*) and pol30(K127R) siz1 show an epistatic relationship. Modifications were analysed as in D.
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gks892-F4: Relevance of the SIM–SUMO interaction for Rad18 function in vivo. (A) Damage-induced PCNA ubiquitylation is reduced in the SIM* mutant. HisPCNA was isolated by Ni–NTA pull-down under denaturing conditions from extracts of MMS-treated cells, and ubiquitin conjugates were detected by anti-ubiquitin western blot. (B) Mutation of the SIM confers enhanced sensitivity to MMS in a manner dependent on K164 of PCNA, but the phenotype is suppressed by deletion of SIZ1. Deletion mutants of rad18 were complemented with RAD18 (WT or SIM*) for spot assays. An empty vector served as control. (C) Mutation of the SIM confers enhanced UV sensitivity. (D) Damage-induced PCNA ubiquitylation is reduced in a mutant deficient in PCNA sumoylation, pol30(K127R) siz1. A modification time course was analysed as in panel A. (E) Quantification of the blots shown in panel D, relative to the WT signal at 60 min. (F) The pol30(K127R) siz1 mutant displays enhanced UV sensitivity. (G) The PCNA ubiquitylation defects of rad18(SIM*) and pol30(K127R) siz1 show an epistatic relationship. Modifications were analysed as in D.

Mentions: Consistent with a relevance of the SIM for Rad18 function in vivo, damage-induced PCNA ubiquitylation was reduced in the mutant (Figure 4A), and the cells exhibited moderate sensitivity towards the alkylating agent MMS and UV radiation (Figure 4B and C). Mutation of K164 of PCNA abolished this effect, confirming that the damage sensitivity is specific to a defect in PCNA ubiquitylation (Figure 4B). Deletion of SIZ1 suppressed the phenotype of rad18(SIM*), as expected from the notion that a failure to recruit Srs2 allows damage processing by homologous recombination. The partial loss of function was not likely caused by a destabilization of the protein, as equal levels of wild-type and mutant Rad189myc were detectable in total cell extracts (Figure 2A). Yet, despite a WT-like activity towards native PCNA in vitro (Figure 3F), we had observed a reduced interaction of the mutated protein with PCNA (Figures 1B and 2D). To rule out that this reduction in PCNA binding was causing the phenotype of the mutant in vivo, we examined a strain bearing WT RAD18, but devoid of PCNA sumoylation, pol30(K127R) siz1. In this strain, PCNA ubiquitylation was also reduced >3-fold (Figure 4D and E), and cells again exhibited UV sensitivity (Figure 4F). Combination of these mutations with rad18(SIM*) did not lead to a loss of PCNA ubiquitylation beyond the level of the rad18(SIM*) single mutant, demonstrating an epistatic relationship between the two defects (Figure 4G). Hence, a functional SIM–SUMO interaction contributes to full activity of Rad18 towards PCNA, and its loss gives rise to a phenotype comparable with that of mutants in other components of the pathway, such as rev3 or rad30 (15).Figure 4.


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

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

Relevance of the SIM–SUMO interaction for Rad18 function in vivo. (A) Damage-induced PCNA ubiquitylation is reduced in the SIM* mutant. HisPCNA was isolated by Ni–NTA pull-down under denaturing conditions from extracts of MMS-treated cells, and ubiquitin conjugates were detected by anti-ubiquitin western blot. (B) Mutation of the SIM confers enhanced sensitivity to MMS in a manner dependent on K164 of PCNA, but the phenotype is suppressed by deletion of SIZ1. Deletion mutants of rad18 were complemented with RAD18 (WT or SIM*) for spot assays. An empty vector served as control. (C) Mutation of the SIM confers enhanced UV sensitivity. (D) Damage-induced PCNA ubiquitylation is reduced in a mutant deficient in PCNA sumoylation, pol30(K127R) siz1. A modification time course was analysed as in panel A. (E) Quantification of the blots shown in panel D, relative to the WT signal at 60 min. (F) The pol30(K127R) siz1 mutant displays enhanced UV sensitivity. (G) The PCNA ubiquitylation defects of rad18(SIM*) and pol30(K127R) siz1 show an epistatic relationship. Modifications were analysed as in D.
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Related In: Results  -  Collection

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gks892-F4: Relevance of the SIM–SUMO interaction for Rad18 function in vivo. (A) Damage-induced PCNA ubiquitylation is reduced in the SIM* mutant. HisPCNA was isolated by Ni–NTA pull-down under denaturing conditions from extracts of MMS-treated cells, and ubiquitin conjugates were detected by anti-ubiquitin western blot. (B) Mutation of the SIM confers enhanced sensitivity to MMS in a manner dependent on K164 of PCNA, but the phenotype is suppressed by deletion of SIZ1. Deletion mutants of rad18 were complemented with RAD18 (WT or SIM*) for spot assays. An empty vector served as control. (C) Mutation of the SIM confers enhanced UV sensitivity. (D) Damage-induced PCNA ubiquitylation is reduced in a mutant deficient in PCNA sumoylation, pol30(K127R) siz1. A modification time course was analysed as in panel A. (E) Quantification of the blots shown in panel D, relative to the WT signal at 60 min. (F) The pol30(K127R) siz1 mutant displays enhanced UV sensitivity. (G) The PCNA ubiquitylation defects of rad18(SIM*) and pol30(K127R) siz1 show an epistatic relationship. Modifications were analysed as in D.
Mentions: Consistent with a relevance of the SIM for Rad18 function in vivo, damage-induced PCNA ubiquitylation was reduced in the mutant (Figure 4A), and the cells exhibited moderate sensitivity towards the alkylating agent MMS and UV radiation (Figure 4B and C). Mutation of K164 of PCNA abolished this effect, confirming that the damage sensitivity is specific to a defect in PCNA ubiquitylation (Figure 4B). Deletion of SIZ1 suppressed the phenotype of rad18(SIM*), as expected from the notion that a failure to recruit Srs2 allows damage processing by homologous recombination. The partial loss of function was not likely caused by a destabilization of the protein, as equal levels of wild-type and mutant Rad189myc were detectable in total cell extracts (Figure 2A). Yet, despite a WT-like activity towards native PCNA in vitro (Figure 3F), we had observed a reduced interaction of the mutated protein with PCNA (Figures 1B and 2D). To rule out that this reduction in PCNA binding was causing the phenotype of the mutant in vivo, we examined a strain bearing WT RAD18, but devoid of PCNA sumoylation, pol30(K127R) siz1. In this strain, PCNA ubiquitylation was also reduced >3-fold (Figure 4D and E), and cells again exhibited UV sensitivity (Figure 4F). Combination of these mutations with rad18(SIM*) did not lead to a loss of PCNA ubiquitylation beyond the level of the rad18(SIM*) single mutant, demonstrating an epistatic relationship between the two defects (Figure 4G). Hence, a functional SIM–SUMO interaction contributes to full activity of Rad18 towards PCNA, and its loss gives rise to a phenotype comparable with that of mutants in other components of the pathway, such as rev3 or rad30 (15).Figure 4.

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