Tadahide Izumi

Apurinic/apyrimidinic endonuclease-1 (APE1) is a multifunctional DNA repair/gene regulatory protein in mammalian cells, and was recently reported to be phosphorylated at Thr233 by CDK5. We here report that ubiquitination of T233E APE1, a mimicry of phospho-T233 APE1, was markedly increased in multiple cell lines. Expression of CDK5 enhanced monoubiquitination of endogenous APE1. Polyubiquitinated APE1 was decreased when K48R ubiquitin was expressed, suggesting that polyubiquitination was mediated mainly through Lys48 of ubiquitin. The ubiquitination activity of MDM2, consistent in its role for APE1 ubiquitination, was increased for T233E APE1 compared to the wild-type APE1. In mouse embryonic fibroblasts lacking the MDM2 gene, ubiquitination of T233E APE1 was still observed probably because of the decreased degradation activity for monoubiquitinated APE1 and because of backup E3 ligases in the cells. Monoubiquitinated APE1 was present in the nucleus, and analyzing global gene expression profiles with or without induction of a ubiquitin-APE1 fusion gene suggested that monoubiquitination enhanced the gene suppression activity of APE1. These data reveal a delicate balance of ubiquitination and phosphorylation activities that alter the gene regulatory function of APE1.

A key issue in studying mammalian DNA base excision repair is how its component proteins respond to a plethora of cell-signaling mediators invoked by DNA damage and stress-inducing agents such as reactive oxygen species, and how the actions of individual BER proteins are attributed to cell survival or apoptotic/necrotic death. This article reviews the past and recent progress on posttranslational modification (PTM) of mammalian apurinic/apyrimidinic (AP) endonuclease 1 (APE1).

APE1/Ref-1 is an essential DNA repair/gene regulatory protein in mammals of which intracellular level significantly affects cellular sensitivity to genotoxicants. The apurinic/apyrimidinic endonuclease 1 (APE1) functions are altered by phosphorylation and acetylation. We here report that APE1 is also modified by ubiquitination. APE1 ubiquitination occurred specifically at Lys residues near the N-terminus, and was markedly enhanced by mouse double minute 2 (MDM2), the major intracellular p53 inhibitor. Moreover, DNA-damaging reagents and nutlin-3, an inhibitor of MDM2–p53 interaction, increased APE1 ubiquitination in the presence of p53. Downmodulation of MDM2 increased APE1 level, suggesting that MDM2-mediated ubiquitination can be a signal for APE1 degradation. In addition, unlike the wild-type APE1, ubiquitin–APE1 fusion proteins were predominantly present in the cytoplasm. Therefore, monoubiquitination not only is a prerequisite for degradation, but may also alter the APE1 activities in cells. These results reveal a novel regulation of APE1 through ubiquitination.

Apurinic/apyrimidinic endonuclease (APE‐1) is essential for base excision repair (BER) of damaged DNA. Here molecular dynamics (MD) simulations of APE1 complexed with cleaved and uncleaved damaged DNA were used to determine the role and position of the metal ion(s) in the active site before and after DNA cleavage. The simulations started from an energy minimized wild‐type structure of the metal‐free APE1/damaged‐DNA complex (1DE8). A grid search with one Mg²⁺ ion located two low energy clusters of Mg²⁺ consistent with the experimentally determined metal ion positions. At the start of the longer MD simulations, Mg²⁺ ions were placed at different positions as seen in the crystal structures and the movement of the ion was followed over the course of the trajectory. Our analysis suggests a “moving metal mechanism” in which one Mg²⁺ ion moves from the B‐ (more buried) to the A‐site during substrate cleavage. The anticipated inversion of the phosphate oxygens occurs during the in‐line cleavage reaction. Experimental results, which show competition between Ca²⁺ and Mg²⁺ for catalyzing the reaction, and high concentrations of Mg²⁺ are inihibitory, indicate that both sites cannot be simultaneously occupied for maximal activity. Proteins 2007.

The mammalian abasic-endonuclease1/redox-factor1 (APE1/Ref1) is an essential protein whose subcellular distribution depends on the cellular physiological status. However, its nuclear localization signals have not been studied in detail. We examined nuclear translocation of APE1, by monitoring enhanced green fluorescent protein (EGFP) fused to APE1. APE1's nuclear localization was significantly decreased by deleting 20 amino acid residues from its N-terminus. Fusion of APE1's N-terminal 20 residues directed nuclear localization of EGFP. An APE1 mutant lacking the seven N-terminal residues (ND7 APE1) showed nearly normal nuclear localization, which was drastically reduced when the deletion was combined with the E12A/D13A double mutation. On the other hand, nearly normal nuclear localization of the full-length E12A/D13A mutant suggests that the first 7 residues and residues 8–13 can independently promote nuclear import. Both far-western analyses and immuno-pull-down assays indicate interaction of APE1 with karyopherin alpha 1 and 2, which requires the 20 N-terminal residues and implicates nuclear importins in APE1's nuclear translocation. Nuclear accumulation of the ND7 APE1(E12A/D13A) mutant after treatment with the nuclear export inhibitor leptomycin B suggests the presence of a previously unidentified nuclear export signal, and the subcellular distribution of APE1 may be regulated by both nuclear import and export.