Acetylation Regulates WRN Catalytic Activities and Affects Base Excision DNA Repair

Abstract

BackgroundThe Werner protein (WRN), defective in the premature aging disorder Werner syndrome, participates in a number of DNA metabolic processes, and we have been interested in the possible regulation of its function in DNA repair by post-translational modifications. Acetylation mediated by histone acetyltransferases is of key interest because of its potential importance in aging, DNA repair and transcription.Methodology/Principal FindingsHere, we have investigated the p300 acetylation mediated changes on the function of WRN in base excision DNA repair (BER). We show that acetylation of WRN increases in cells treated with methyl methanesulfonate (MMS), suggesting that acetylation of WRN may play a role in response to DNA damage. This hypothesis is consistent with our findings that acetylation of WRN stimulates its catalytic activities in vitro and in vivo, and that acetylated WRN enhances pol β-mediated strand displacement DNA synthesis more than unacetylated WRN. Furthermore, we show that cellular exposure to the histone deacetylase inhibitor sodium butyrate stimulates long patch BER in wild type cells but not in WRN depleted cells, suggesting that acetylated WRN participates significantly in this process.Conclusion/SignificanceCollectively, these results provide the first evidence for a specific role of p300 mediated WRN acetylation in regulating its function during BER.

Acetylation regulates DNA repair mechanisms in human cells

“…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.…”

“…Whereas previous studies showed that LP-BER reactions in vitro were stimulated when performed with whole cells extracts prepared from cells exposed to sodium butyrate, we did not observe such stimulation in our cell-based system. 15 Also, p300-mediated acetylation of different factors involved in BER showed stimulation of synthesis-driven displacement utilized by LP-BER, whereas in our cell-based system depletion of p300 did not affect LP-BER. 15,17,18,43 Specifically, there was no evident effect of acetylation on repair of a mismatch downstream of an 8-oxoG damaged base (GO: MC6).…”

“…15 Also, p300-mediated acetylation of different factors involved in BER showed stimulation of synthesis-driven displacement utilized by LP-BER, whereas in our cell-based system depletion of p300 did not affect LP-BER. 15,17,18,43 Specifically, there was no evident effect of acetylation on repair of a mismatch downstream of an 8-oxoG damaged base (GO: MC6). However, the inhibitory effect of garcinol on repair in GO constructs suggests that at least some steps in BER are affected by loss of acetylation.…”

“…14 Moreover, acetylation of the Werner DNA helicase (WRN) stimulated Pol b-mediated strand displacement DNA synthesis in vitro, suggesting up-regulation of the long patch (LP) BER pathway in cells upon acetylation. 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.…”

“…This result is inconsistent with previous reports showing that p300-mediated acetylation increased the efficiency of reconstituted LP-BER. 15 This specific outcome will be addressed in the Discussion.…”