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CBP and p300 acetylate PCNA to link its degradation with nucleotide excision repair synthesis.

Cazzalini O, Sommatis S, Tillhon M, Dutto I, Bachi A, Rapp A, Nardo T, Scovassi AI, Necchi D, Cardoso MC, Stivala LA, Prosperi E - Nucleic Acids Res. (2014)

Bottom Line: The proliferating cell nuclear antigen (PCNA) protein serves as a molecular platform recruiting and coordinating the activity of factors involved in multiple deoxyribonucleic acid (DNA) transactions.To avoid dangerous genome instability, it is necessary to prevent excessive retention of PCNA on chromatin.Depletion of both CBP and p300, or failure to load PCNA on DNA in NER deficient cells, prevented PCNA acetylation and degradation, while proteasome inhibition resulted in accumulation of acetylated PCNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, University of Pavia, Pavia 27100, Italy.

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PCNA degradation after DNA damage is dependent on acetylation. (A) Time course analysis of PCNA levels in the soluble and chromatin-bound pools of PCNA in quiescent, CHX-treated (25 μM) LF-1 fibroblasts after UV-irradiation (10 J/m2). Western blot analysis of PCNA, histone H3, and actin (loading controls), are shown. (B) Densitometric quantification of the soluble form of PCNA in CHX-treated and UV-irradiated (10 J/m2) quiescent LF-1 fibroblasts. PCNA levels were normalized to actin content. Mean values ± s.d. of at least three independent experiments are shown. (C) Western blot analysis of PCNA and actin (loading control) in the soluble and chromatin-bound pools in LF-1 fibroblasts incubated with non-targeting (NT) siRNA, or siRNA targeting CBP and p300 (CBP/p300), at 4 h after UV irradiation (10 J/m2). (D) Densitometric quantification of soluble PCNA (normalized to actin) in LF-1 fibroblasts incubated with non-targeting siRNA (NT), both siRNA to CBP/p300, or in single exposure, or to PCAF, in untreated (C), or 4 h after UV irradiation (10 J/m2). Mean values ± s.d. of three independent experiments are shown. (E) Western blot analysis of soluble RFP-PCNA and actin (loading control) in HeLa cells expressing wt, 2KR or 5KR mutant RFP-PCNA, before (0), or at 4 and 8 h after UV irradiation (10 J/m2) in the presence of CHX. (F) Densitometric quantification of soluble RFP-PCNA levels (normalized to actin) at 4 h after UV irradiation (10 J/m2) in HeLa cells expressing wt, 2KR or 5KR RFP-PCNA. Mean values ± s.d. of three independent experiments are shown. (G) Immunoprecipitation of RFP-PCNA with anti-RFP antibody in whole extracts from HeLa cells expressing wt, 2KR or 5KR RFP-PCNA, at 30 min after UV irradiation (10 J/m2), in the presence of MG132. Western blot analysis of immunocomplexes was performed with anti-ubiquitin antibody. RFP-PCNA levels in the input extracts are shown below.
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Figure 7: PCNA degradation after DNA damage is dependent on acetylation. (A) Time course analysis of PCNA levels in the soluble and chromatin-bound pools of PCNA in quiescent, CHX-treated (25 μM) LF-1 fibroblasts after UV-irradiation (10 J/m2). Western blot analysis of PCNA, histone H3, and actin (loading controls), are shown. (B) Densitometric quantification of the soluble form of PCNA in CHX-treated and UV-irradiated (10 J/m2) quiescent LF-1 fibroblasts. PCNA levels were normalized to actin content. Mean values ± s.d. of at least three independent experiments are shown. (C) Western blot analysis of PCNA and actin (loading control) in the soluble and chromatin-bound pools in LF-1 fibroblasts incubated with non-targeting (NT) siRNA, or siRNA targeting CBP and p300 (CBP/p300), at 4 h after UV irradiation (10 J/m2). (D) Densitometric quantification of soluble PCNA (normalized to actin) in LF-1 fibroblasts incubated with non-targeting siRNA (NT), both siRNA to CBP/p300, or in single exposure, or to PCAF, in untreated (C), or 4 h after UV irradiation (10 J/m2). Mean values ± s.d. of three independent experiments are shown. (E) Western blot analysis of soluble RFP-PCNA and actin (loading control) in HeLa cells expressing wt, 2KR or 5KR mutant RFP-PCNA, before (0), or at 4 and 8 h after UV irradiation (10 J/m2) in the presence of CHX. (F) Densitometric quantification of soluble RFP-PCNA levels (normalized to actin) at 4 h after UV irradiation (10 J/m2) in HeLa cells expressing wt, 2KR or 5KR RFP-PCNA. Mean values ± s.d. of three independent experiments are shown. (G) Immunoprecipitation of RFP-PCNA with anti-RFP antibody in whole extracts from HeLa cells expressing wt, 2KR or 5KR RFP-PCNA, at 30 min after UV irradiation (10 J/m2), in the presence of MG132. Western blot analysis of immunocomplexes was performed with anti-ubiquitin antibody. RFP-PCNA levels in the input extracts are shown below.

Mentions: Next, PCNA turnover was analyzed in LF-1 fibroblasts after UV-irradiation in the presence of CHX to prevent new protein synthesis (Figure 7A). Chromatin-bound PCNA showed the typical accumulation for DNA repair synthesis (62,63), even in the presence of CHX. In contrast, PCNA levels in the soluble fraction underwent a significant reduction, with a half-time of ∼3 h (Figure 7B). Noticeably, PCNA degradation occurred even when DNA damage was induced either by an oxidizing, or alkylating agent, or by ionizing radiation (Supplementary Figure S5B). Analysis of PCNA levels in both detergent-soluble and chromatin-bound fractions showed that degradation of the soluble form was completely rescued, and its level was even increased after depleting both CBP and p300 (Figure 7C). A detectable increase (by about 1.5×) in the amount of soluble form of PCNA was observed in non irradiated cells after siRNA depletion of both CBP and p300, indicating that their activity could influence the stability of PCNA even in unperturbed conditions. In CBP/p300-depleted and UV-irradiated cells, the increase in the soluble form of PCNA was about twice the amount observed in untreated control cells (Figure 7D). The dependence of PCNA degradation on the activity of each enzyme was also investigated by incubating LF-1 fibroblasts with siRNA to either CBP or p300, before irradiation. The effect of the single depletion was not significantly different from that observed after co-depletion of both CBP and p300, although the lower levels of soluble PCNA after irradiation suggested that both activities were important for PCNA degradation. In contrast, depletion of PCAF (Supplementary Figure S5C) did not influence UV-induced PCNA degradation (Figure 7D and Supplementary Figure S5D). The dependence of PCNA degradation on the acetylation of specific lysine residues was also verified by monitoring levels of the soluble form of RFP-PCNAwt versus the mutant forms after UV irradiation (Figure 7E). A significant resistance of RFP-PCNA2KR and RFP-PCNA5KR to proteolytic destruction was found after UV-irradiation, while RFP-PCNAwt was degraded (Figure 7F). To prove that acetylation was required for signaling PCNA degradation through ubiquitination, HeLa cells expressing RFP-PCNAwt or mutants were UV-irradiated in the presence of MG132, a proteasome inhibitor. Immunoprecipitation of RFP-PCNA showed that ubiquitinated forms were accumulated in cells expressing RFP-PCNAwt, while they could not be observed in 2KR, or 5KR mutants (Figure 7G). These results suggest that CBP/p300-mediated acetylation is necessary to trigger PCNA proteasomal degradation.


CBP and p300 acetylate PCNA to link its degradation with nucleotide excision repair synthesis.

Cazzalini O, Sommatis S, Tillhon M, Dutto I, Bachi A, Rapp A, Nardo T, Scovassi AI, Necchi D, Cardoso MC, Stivala LA, Prosperi E - Nucleic Acids Res. (2014)

PCNA degradation after DNA damage is dependent on acetylation. (A) Time course analysis of PCNA levels in the soluble and chromatin-bound pools of PCNA in quiescent, CHX-treated (25 μM) LF-1 fibroblasts after UV-irradiation (10 J/m2). Western blot analysis of PCNA, histone H3, and actin (loading controls), are shown. (B) Densitometric quantification of the soluble form of PCNA in CHX-treated and UV-irradiated (10 J/m2) quiescent LF-1 fibroblasts. PCNA levels were normalized to actin content. Mean values ± s.d. of at least three independent experiments are shown. (C) Western blot analysis of PCNA and actin (loading control) in the soluble and chromatin-bound pools in LF-1 fibroblasts incubated with non-targeting (NT) siRNA, or siRNA targeting CBP and p300 (CBP/p300), at 4 h after UV irradiation (10 J/m2). (D) Densitometric quantification of soluble PCNA (normalized to actin) in LF-1 fibroblasts incubated with non-targeting siRNA (NT), both siRNA to CBP/p300, or in single exposure, or to PCAF, in untreated (C), or 4 h after UV irradiation (10 J/m2). Mean values ± s.d. of three independent experiments are shown. (E) Western blot analysis of soluble RFP-PCNA and actin (loading control) in HeLa cells expressing wt, 2KR or 5KR mutant RFP-PCNA, before (0), or at 4 and 8 h after UV irradiation (10 J/m2) in the presence of CHX. (F) Densitometric quantification of soluble RFP-PCNA levels (normalized to actin) at 4 h after UV irradiation (10 J/m2) in HeLa cells expressing wt, 2KR or 5KR RFP-PCNA. Mean values ± s.d. of three independent experiments are shown. (G) Immunoprecipitation of RFP-PCNA with anti-RFP antibody in whole extracts from HeLa cells expressing wt, 2KR or 5KR RFP-PCNA, at 30 min after UV irradiation (10 J/m2), in the presence of MG132. Western blot analysis of immunocomplexes was performed with anti-ubiquitin antibody. RFP-PCNA levels in the input extracts are shown below.
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Figure 7: PCNA degradation after DNA damage is dependent on acetylation. (A) Time course analysis of PCNA levels in the soluble and chromatin-bound pools of PCNA in quiescent, CHX-treated (25 μM) LF-1 fibroblasts after UV-irradiation (10 J/m2). Western blot analysis of PCNA, histone H3, and actin (loading controls), are shown. (B) Densitometric quantification of the soluble form of PCNA in CHX-treated and UV-irradiated (10 J/m2) quiescent LF-1 fibroblasts. PCNA levels were normalized to actin content. Mean values ± s.d. of at least three independent experiments are shown. (C) Western blot analysis of PCNA and actin (loading control) in the soluble and chromatin-bound pools in LF-1 fibroblasts incubated with non-targeting (NT) siRNA, or siRNA targeting CBP and p300 (CBP/p300), at 4 h after UV irradiation (10 J/m2). (D) Densitometric quantification of soluble PCNA (normalized to actin) in LF-1 fibroblasts incubated with non-targeting siRNA (NT), both siRNA to CBP/p300, or in single exposure, or to PCAF, in untreated (C), or 4 h after UV irradiation (10 J/m2). Mean values ± s.d. of three independent experiments are shown. (E) Western blot analysis of soluble RFP-PCNA and actin (loading control) in HeLa cells expressing wt, 2KR or 5KR mutant RFP-PCNA, before (0), or at 4 and 8 h after UV irradiation (10 J/m2) in the presence of CHX. (F) Densitometric quantification of soluble RFP-PCNA levels (normalized to actin) at 4 h after UV irradiation (10 J/m2) in HeLa cells expressing wt, 2KR or 5KR RFP-PCNA. Mean values ± s.d. of three independent experiments are shown. (G) Immunoprecipitation of RFP-PCNA with anti-RFP antibody in whole extracts from HeLa cells expressing wt, 2KR or 5KR RFP-PCNA, at 30 min after UV irradiation (10 J/m2), in the presence of MG132. Western blot analysis of immunocomplexes was performed with anti-ubiquitin antibody. RFP-PCNA levels in the input extracts are shown below.
Mentions: Next, PCNA turnover was analyzed in LF-1 fibroblasts after UV-irradiation in the presence of CHX to prevent new protein synthesis (Figure 7A). Chromatin-bound PCNA showed the typical accumulation for DNA repair synthesis (62,63), even in the presence of CHX. In contrast, PCNA levels in the soluble fraction underwent a significant reduction, with a half-time of ∼3 h (Figure 7B). Noticeably, PCNA degradation occurred even when DNA damage was induced either by an oxidizing, or alkylating agent, or by ionizing radiation (Supplementary Figure S5B). Analysis of PCNA levels in both detergent-soluble and chromatin-bound fractions showed that degradation of the soluble form was completely rescued, and its level was even increased after depleting both CBP and p300 (Figure 7C). A detectable increase (by about 1.5×) in the amount of soluble form of PCNA was observed in non irradiated cells after siRNA depletion of both CBP and p300, indicating that their activity could influence the stability of PCNA even in unperturbed conditions. In CBP/p300-depleted and UV-irradiated cells, the increase in the soluble form of PCNA was about twice the amount observed in untreated control cells (Figure 7D). The dependence of PCNA degradation on the activity of each enzyme was also investigated by incubating LF-1 fibroblasts with siRNA to either CBP or p300, before irradiation. The effect of the single depletion was not significantly different from that observed after co-depletion of both CBP and p300, although the lower levels of soluble PCNA after irradiation suggested that both activities were important for PCNA degradation. In contrast, depletion of PCAF (Supplementary Figure S5C) did not influence UV-induced PCNA degradation (Figure 7D and Supplementary Figure S5D). The dependence of PCNA degradation on the acetylation of specific lysine residues was also verified by monitoring levels of the soluble form of RFP-PCNAwt versus the mutant forms after UV irradiation (Figure 7E). A significant resistance of RFP-PCNA2KR and RFP-PCNA5KR to proteolytic destruction was found after UV-irradiation, while RFP-PCNAwt was degraded (Figure 7F). To prove that acetylation was required for signaling PCNA degradation through ubiquitination, HeLa cells expressing RFP-PCNAwt or mutants were UV-irradiated in the presence of MG132, a proteasome inhibitor. Immunoprecipitation of RFP-PCNA showed that ubiquitinated forms were accumulated in cells expressing RFP-PCNAwt, while they could not be observed in 2KR, or 5KR mutants (Figure 7G). These results suggest that CBP/p300-mediated acetylation is necessary to trigger PCNA proteasomal degradation.

Bottom Line: The proliferating cell nuclear antigen (PCNA) protein serves as a molecular platform recruiting and coordinating the activity of factors involved in multiple deoxyribonucleic acid (DNA) transactions.To avoid dangerous genome instability, it is necessary to prevent excessive retention of PCNA on chromatin.Depletion of both CBP and p300, or failure to load PCNA on DNA in NER deficient cells, prevented PCNA acetylation and degradation, while proteasome inhibition resulted in accumulation of acetylated PCNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, University of Pavia, Pavia 27100, Italy.

Show MeSH
Related in: MedlinePlus