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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 acetylation mutants interact with DNA polymerase δ and CBP/p300. (A) Immunoprecipitation of RFP-PCNA wt and mutant forms with anti-RFP antibody. Immunocomplexes were analyzed by western blot with antibodies to RFP and DNA polymerase δ (p125). (B) Recruitment of RFP-PCNAwt, RFP-PCNA2KR and RFP-PCNA5KR with DNA polymerase δ (p125) to sites of local damage 30 min after UV irradiation (30 J/m2). Cells were stained with anti-p125 antibody and analyzed by confocal microscopy. Merged images show co-localization on of RFP-PCNA, p125 and DNA stained with Hoechst 33258. Bar = 10 μm. (C) Immunoprecipitation of RFP-PCNA wt and mutant forms with antibodies to CBP or p300. Immunocomplexes were analyzed by western blot with antibodies to CBP, p300, RFP and PCNA.
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Figure 5: PCNA acetylation mutants interact with DNA polymerase δ and CBP/p300. (A) Immunoprecipitation of RFP-PCNA wt and mutant forms with anti-RFP antibody. Immunocomplexes were analyzed by western blot with antibodies to RFP and DNA polymerase δ (p125). (B) Recruitment of RFP-PCNAwt, RFP-PCNA2KR and RFP-PCNA5KR with DNA polymerase δ (p125) to sites of local damage 30 min after UV irradiation (30 J/m2). Cells were stained with anti-p125 antibody and analyzed by confocal microscopy. Merged images show co-localization on of RFP-PCNA, p125 and DNA stained with Hoechst 33258. Bar = 10 μm. (C) Immunoprecipitation of RFP-PCNA wt and mutant forms with antibodies to CBP or p300. Immunocomplexes were analyzed by western blot with antibodies to CBP, p300, RFP and PCNA.

Mentions: Since lysine mutations in PCNA affect DNA polymerase δ activity (58), we investigated whether our mutants retained the ability to interact with this polymerase, by immunoprecipitation with anti-RFP antibody. DNA polymerase δ (p125 subunit) co-immunoprecipitated with RFP-PCNAwt, as well as with 2KR and 5KR mutants, both in the soluble and in the chromatin-bound fractions (Figure 5A). In the latter extract, interaction with PCNA mutants appeared to be more stable than that with RFP-PCNAwt. To verify that DNA polymerase δ was also relocated to sites of DNA damage together with PCNA, HeLa cells expressing RFP-PCNAwt, 2KR or 5KR mutants were locally UV-irradiated and analyzed for protein recruitment. Confocal microscopy analysis of RFP fluorescence together with immunofluorescence labeling showed that DNA polymerase δ co-localized with PCNAwt and mutant forms, to sites of DNA damage (Figure 5B).


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 acetylation mutants interact with DNA polymerase δ and CBP/p300. (A) Immunoprecipitation of RFP-PCNA wt and mutant forms with anti-RFP antibody. Immunocomplexes were analyzed by western blot with antibodies to RFP and DNA polymerase δ (p125). (B) Recruitment of RFP-PCNAwt, RFP-PCNA2KR and RFP-PCNA5KR with DNA polymerase δ (p125) to sites of local damage 30 min after UV irradiation (30 J/m2). Cells were stained with anti-p125 antibody and analyzed by confocal microscopy. Merged images show co-localization on of RFP-PCNA, p125 and DNA stained with Hoechst 33258. Bar = 10 μm. (C) Immunoprecipitation of RFP-PCNA wt and mutant forms with antibodies to CBP or p300. Immunocomplexes were analyzed by western blot with antibodies to CBP, p300, RFP and PCNA.
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Figure 5: PCNA acetylation mutants interact with DNA polymerase δ and CBP/p300. (A) Immunoprecipitation of RFP-PCNA wt and mutant forms with anti-RFP antibody. Immunocomplexes were analyzed by western blot with antibodies to RFP and DNA polymerase δ (p125). (B) Recruitment of RFP-PCNAwt, RFP-PCNA2KR and RFP-PCNA5KR with DNA polymerase δ (p125) to sites of local damage 30 min after UV irradiation (30 J/m2). Cells were stained with anti-p125 antibody and analyzed by confocal microscopy. Merged images show co-localization on of RFP-PCNA, p125 and DNA stained with Hoechst 33258. Bar = 10 μm. (C) Immunoprecipitation of RFP-PCNA wt and mutant forms with antibodies to CBP or p300. Immunocomplexes were analyzed by western blot with antibodies to CBP, p300, RFP and PCNA.
Mentions: Since lysine mutations in PCNA affect DNA polymerase δ activity (58), we investigated whether our mutants retained the ability to interact with this polymerase, by immunoprecipitation with anti-RFP antibody. DNA polymerase δ (p125 subunit) co-immunoprecipitated with RFP-PCNAwt, as well as with 2KR and 5KR mutants, both in the soluble and in the chromatin-bound fractions (Figure 5A). In the latter extract, interaction with PCNA mutants appeared to be more stable than that with RFP-PCNAwt. To verify that DNA polymerase δ was also relocated to sites of DNA damage together with PCNA, HeLa cells expressing RFP-PCNAwt, 2KR or 5KR mutants were locally UV-irradiated and analyzed for protein recruitment. Confocal microscopy analysis of RFP fluorescence together with immunofluorescence labeling showed that DNA polymerase δ co-localized with PCNAwt and mutant forms, to sites of DNA damage (Figure 5B).

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