The Ape1-NPM1 Axis and Telomere Maintenance



 DESCRIPTION (provided by applicant): Telomeres require special mechanisms for their maintenance, and disruption of these mechanisms, or of the telomeric structures themselves, occur in nearly all cancers. Damage to telomeric DNA disrupts telomere maintenance and can result in chromosome rearrangements. We found recently that Ape1 protein, the central player of base excision DNA repair, is required for proper telomere maintenance in both normal mammalian cells and in tumor lines. Ape1 is required for normal binding of the protective protein TRF2, and Ape1 deficiency leads to increased telomeric binding of POT1. The AP endonuclease activity (DNA repair function) of Ape1 is required for telomere maintenance. Although Ape1 is found throughout the nucleus, it accumulates preferentially in the nucleolus via interaction with nucleophosmin (NPM1), from which it is released in DNA-damaged cells through acetylation of lysines near the Ape1 N- terminus. While DNA damage and telomere disruption are mechanistically related by Ape1, it is unknown how the modulation of Ape1 by NPM1 plays a role in this process. Moreover, the NPM1c+ mutations that occur in about 1/3 of acute myelogenous leukemias (AML) lead to relocation of NPM1 to the cytoplasm, carrying Ape1 with it. In this revised application, we will examine the effects of the NPM1-Ape1 interaction on telomere maintenance, and its possible role in the etiology of AML and other cancers. Although Ape1 cannot be eliminated, we have established RNAi protocols for its efficient down-regulation. The interaction with NPM1 can be controlled by replacement of the key N-terminal lysines (27, 31, 32, 35) of Ape1 with alanines (to mimic the uncharged acetylated state) or arginines (to prevent actylation and retain charge). These tools, and cells expressing the NPM1c+ protein, will be used to determine whether the Ape1-NPM1 interaction is important for telomere maintenance under both normal conditions, and in the face of DNA damage by oxidative or alkylating agents, which generate lesions processed by base excision repair. Telomere length, and the occupation by TRF2 protein, will be monitored by methods we have already established.