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Conformational Rearrangements Regulating the DNA Repair Protein APE1

ABSTRACT: Apurinic apyrimidinic endonuclease 1 (APE1) is a key enzyme of the Base Excision Repair (BER) pathway, which primarily manages oxidative lesions of DNA. Once the damaged base is removed, APE1 recognises the resulting abasic site and cleaves the phosphodiester backbone to allow for the correction by subsequent enzymes of the BER machinery. In spite of a wealth of information on APE1 structure and activity, its regulation mechanism still remains to be understood. Human APE1 consists of a globular catalytic domain preceded by a flexible N-terminal extension, which might be involved in the interaction with DNA. Moreover, the binding of the nuclear chaperone nucleophosmin (NPM1) to this region has been reported to impact APE1 catalysis. To evaluate intra- and inter-molecular conformational rearrangements upon DNA binding, incision, and interaction with NPM1, we used Förster resonance energy transfer (FRET), a fluorescence spectroscopy technique sensitive to molecular distances. Our results suggest that the N-terminus approaches the DNA at the downstream side of the abasic site and enables the building of a predictive model of the full-length APE1/DNA complex. Furthermore, the spatial configuration of the N-terminal tail is sensitive to NPM1, which could be related to the regulation of APE1.

SUBMITTER: Komaniecka N

PROVIDER: S-EPMC9324194 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Although chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect of platinum drugs, the mechanisms of this toxicity remain unknown. Previous work in our laboratory suggests that cisplatin-induced CIPN is secondary to DNA damage which is susceptible to base excision repair (BER). To further examine this hypothesis, we studied the effects of cisplatin, oxaliplatin, and carboplatin on cell survival, DNA damage, ROS production, and functional endpoints in rat sensory neurons in culture in the absence or presence of reduced expression of the BER protein AP endonuclease/redox factor-1 (APE1). Using an in situ model of peptidergic sensory neuron function, we examined the effects of the platinum drugs on hind limb capsaicin-evoked vasodilatation. Exposing sensory neurons in culture to the three platinum drugs caused a concentration-dependent increase in apoptosis and cell death, although the concentrations of carboplatin were 10 fold higher than cisplatin. As previously observed with cisplatin, oxaliplatin and carboplatin also increased DNA damage as indicated by an increase in phospho-H2AX and reduced the capsaicin-evoked release of CGRP from neuronal cultures. Both cisplatin and oxaliplatin increased the production of ROS as well as 8-oxoguanine DNA adduct levels, whereas carboplatin did not. Reducing levels of APE1 in neuronal cultures augmented the cisplatin and oxaliplatin induced toxicity, but did not alter the effects of carboplatin. Using an in vivo model, systemic injection of cisplatin (3 mg/kg), oxaliplatin (3 mg/kg), or carboplatin (30 mg/kg) once a week for three weeks caused a decrease in capsaicin-evoked vasodilatation, which was delayed in onset. The effects of cisplatin on capsaicin-evoked vasodilatation were attenuated by chronic administration of E3330, a redox inhibitor of APE1 that serendipitously enhances APE1 DNA repair activity in sensory neurons. These outcomes support the importance of the BER pathway, and particularly APE1, in sensory neuropathy caused by cisplatin and oxaliplatin, but not carboplatin and suggest that augmenting DNA repair could be a therapeutic target for CIPN.

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Conformational Rearrangements Regulating the DNA Repair Protein APE1

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