CBP and p300 acetylate PCNA to link its degradation with nucleotide excision repair synthesis.
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.
Affiliation: Department of Molecular Medicine, University of Pavia, Pavia 27100, Italy.Show MeSH
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Mentions: Since lysine mutations in PCNA affect DNA polymerase Î´ activity (58), we investigated whether our mutants retained the ability to interact with this polymerase, by immunoprecipitation with anti-RFP antibody. DNA polymerase Î´ (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 Î´ 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 Î´ co-localized with PCNAwt and mutant forms, to sites of DNA damage (Figure 5B).
Affiliation: Department of Molecular Medicine, University of Pavia, Pavia 27100, Italy.