The Post-translational Modifications of Proliferating Cell Nuclear Antigen

Abstract

The diverse function of proliferating cell nuclear antigen (PCNA) is thought to be due, in large part, to posttranslational modifications. Here we show by high resolution two-dimensional PAGE analysis that there are three distinct PCNA isoforms that differ in their acetylation status. The moderately acetylated main (M) form was found in all of the subcellular compartments of cycling cells, whereas the highly acetylated acidic form was primarily found in the nucleoplasm, nuclear matrix, and chromatin. Interestingly, the deacetylated basic form was most pronounced in the nucleoplasm of cycling cells. The cells in G 0 and the cytoplasm of cycling cells contained primarily the M form only. Because p300 and histone deacetylase (HDAC1) were co-immunoprecipitated with PCNA, they are likely responsible for the acetylation and deacetylation of PCNA, respectively. We also found that deacetylation reduced the ability of PCNA to bind to DNA polymerases ␤ and ␦. Taken together, our data support a model where the acidic and M forms participate in DNA replication, whereas the basic form is associated with the termination of DNA replication. PCNA1 participates in a broad spectrum of biological activities, including DNA replication, repair, cell cycle control, apoptosis, and chromatin remodeling (1-3). Several reports (4 -7) previously suggested the existence of different PCNA species, which is thought to be relevant to the diverse functions of PCNA. Furthermore, we have shown recently by high resolution two-dimensional PAGE and immunoblot analyses that both hamster and human cells contain three different PCNA isoforms, the acidic (A), the main (M), and the basic (B) forms (8). The different PCNA isoforms could be the result of posttranslational modifications, especially phosphorylation status (9 -12). Consistent with this hypothesis, Prosperi et al. (13) presented data that support the phosphorylation of PCNA. In contrast, however, Bravo and Celis (14) did not find any evidence of PCNA phosphorylation. We wanted to address this issue by carrying out several different experimental approaches, including in vivo labeling, immunoprecipitation, and immunoblot analysis in combination with high resolution twodimensional PAGE. Here we show that mammalian PCNA is regulated not by phosphorylation but by acetylation. We have also shown that three distinct PCNA isoforms differ in their acetylation status. Our data further show that acetylation is associated with subcellular localization of PCNA. Moreover, deacetylated PCNA by HDAC1 has lower affinity to DNA polymerases ␤ and ␦ than that of PCNA treated with trichostatin A (TSA, an HDAC inhibitor). Most important, the PCNA-DNA polymerase complex treated with HDAC1 showed lower polymerization activity than a TSA-treated control. Taken together, our data are consistent with the idea that acetylated PCNA is involved in the DNA replication process, whereas deacetylation of PCNA is associated with the termination of DNA replication. MATERIALS AND METHODSReagents-All the reagents used are from ...

Acetylation regulates DNA repair mechanisms in human cells

“…The negative effect of p300 loss on mismatch repair in this setting is consistent with results showing that acetylation improves strand displacement. [16][17][18]53 The implication of our results is that the acetylation-dependent increase in patch size observed for Okazaki fragment maturation in vitro, also occurs in cells. We conclude that the nickdependent repair of mismatches and the 5 0 -end processing of Okazaki fragments are improved by protein acetylationdependent increases in strand displacement synthesis.…”

“…In our current work, we approached the experiments knowing that numerous DNA replication and repair proteins are acetylated by p300 and that acetylation changes their properties in a way that should promote more efficient strand displacement and consequently more effective repair. 15,17,18,43,51 In our studies, exposing cells to sodium butyrate (deacetylase inhibitor) produced an increase in repair of nicked plasmid DNA in HeLa cells, and also increases in repair of a mismatch and nicks in HeLa and MMR-deficient HCT116 cells. Exposing of cells to garcinol (acetyltransferase inhibitor) inhibited repair of the diagnostic nicksCmismatch-containing plasmid in both cell lines, and also repair of nicked DNA, but only at higher garcinol concentrations in HCT116 cells.…”

“…p300 was previously indicated as a potential acetyltransferase able to modify BER pathway proteins, including Pol b, OGG1, RPA, PCNA and FEN1. 14,17,18,38,43 As shown in Fig. 7, no differences were observed in relative EGFP levels for the 8-oxoG containing plasmid (GO), as well as for GO:MC6, for both HCT116 cell lines.…”

“…52 Also, enzymes involved in Okazaki fragment maturation are influenced by acetylation to promote strand displacement synthesis. [16][17][18]53 Assessed in vitro, FEN1 had previously been reported to be inhibited approximately about 90% by acetylation. 18 This property would delay ligation of the fragments.…”

“…15 A positive effect of acetylation on strand displacement synthesis was also observed in reconstituted systems simulating Okazaki fragments synthesis. [16][17][18] The effect of acetylation on proteins involved in DNA mismatch repair (MMR) was investigated in less detail. Here, acetylation of K residues at the C termini of MSH2, an especial component of the complex recognizing mismatches in DNA, had a positive effect on repair.…”