Nucleolar accumulation of APE1 depends on charged lysine residues that undergo acetylation upon genotoxic stress and modulate its BER activity in cells

Abstract

The functional importance of APE1 nucleolar accumulation is described. It is shown that acetylation of Lys27–35, affecting local conformation, regulates APE1 function by 1) controlling its interaction with NPM1 and rRNA and its nucleolar accumulation, 2) modulating K6/K7 acetylation status, and 3) promoting APE1 BER activity in cells.

Nucleolin and nucleophosmin: nucleolar proteins with multiple functions in DNA repair

“…Furthermore, they reported that SIRT1 activity mediated the increased association of APE1 with XRCC1, a key BER scaffolding enzyme, in response to MMS treatment, and that depletion of SIRT1 resulted in sensitivity of HeLa cells to MMS and increased AP-site accumulation, both of which could be rescued by overexpression of APE1 (Yamamori et al 2010). However, subsequent work found that SIRT1 interacted with APE1 K4pleR but not with the acetylation-mimicking APE1 K4pleA mutant that is released from the nucleolus by NPM1 upon induction of genotoxic stress, and failed to deacetylate APE1 K4pleA at K6/K7, despite the fact that this form possesses greater AP-endonuclease activity than either APE1 WT or APE1 K4pleR (Lirussi et al 2012). …”

“…This interaction, mediated by binding of the lysine-rich APE1 N-terminal tail to NPM1's N-terminal region containing the oligomerization domain and acidic tracts, results in the robust recruitment of APE1 into nucleoli (Fantini et al 2010;Vascotto et al 2009). Upon induction of genotoxic stress such as base D r a f t 23 oxidation or methylation, lysines within the APE1 N-terminal become acetylated, interrupting the NPM1-APE1 interactions and inducing translocation of APE1 from the nucleolus to the nucleoplasm (Fantini et al 2010;Lirussi et al 2012). Mutation of four of these residues (K27, K31, K32, K35) to acetylation-mimicking uncharged alanines (termed APE1 K4pleA ) results in constitutive translocation of APE1 to the nucleoplasm, while inhibition of acetylation by lysine-to-arginine mutations (APE1 K4pleR ) constitutively prevents translocation; these same mutations inhibit (APE1 K4pleA ) or enhance (APE1 K4pleR ) the interaction of APE1 with NPM1, emphasizing the importance of this interaction to the regulation of APE1's nucleolar sequestration (Lirussi et al 2012).…”

“…Upon induction of genotoxic stress such as base D r a f t 23 oxidation or methylation, lysines within the APE1 N-terminal become acetylated, interrupting the NPM1-APE1 interactions and inducing translocation of APE1 from the nucleolus to the nucleoplasm (Fantini et al 2010;Lirussi et al 2012). Mutation of four of these residues (K27, K31, K32, K35) to acetylation-mimicking uncharged alanines (termed APE1 K4pleA ) results in constitutive translocation of APE1 to the nucleoplasm, while inhibition of acetylation by lysine-to-arginine mutations (APE1 K4pleR ) constitutively prevents translocation; these same mutations inhibit (APE1 K4pleA ) or enhance (APE1 K4pleR ) the interaction of APE1 with NPM1, emphasizing the importance of this interaction to the regulation of APE1's nucleolar sequestration (Lirussi et al 2012). Notably, the APE1 K4pleA mutation shows significantly greater endonuclease activity on AP-DNA and its expression in vivo reduces AP site accumulation, suggesting that sequestration into the nucleolus by NPM1 prevents APE1 action within the BER pathway (Lirussi et al 2012).…”

“…Mutation of four of these residues (K27, K31, K32, K35) to acetylation-mimicking uncharged alanines (termed APE1 K4pleA ) results in constitutive translocation of APE1 to the nucleoplasm, while inhibition of acetylation by lysine-to-arginine mutations (APE1 K4pleR ) constitutively prevents translocation; these same mutations inhibit (APE1 K4pleA ) or enhance (APE1 K4pleR ) the interaction of APE1 with NPM1, emphasizing the importance of this interaction to the regulation of APE1's nucleolar sequestration (Lirussi et al 2012). Notably, the APE1 K4pleA mutation shows significantly greater endonuclease activity on AP-DNA and its expression in vivo reduces AP site accumulation, suggesting that sequestration into the nucleolus by NPM1 prevents APE1 action within the BER pathway (Lirussi et al 2012). Several other BER proteins including FEN1 and Ligase III are also reported to exhibit NPM1-dependent nucleolar localization, which may be reversed following genotoxic stress, suggesting that this mechanism may control multiple steps of the BER repair pathway ).…”

“…The first is that, while APE1 is enriched in the nucleoli, extensive nucleoplasmic APE1 staining is still observed in most cells, suggesting that NPM1 sequesters only a relatively minor pool (an estimated 5-10% of total cellular APE1) into nucleoli (Lirussi et al 2012;Vascotto et al 2009). …”