Abstract
Apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is the primary enzyme in mammals for the repair of abasic sites in DNA, as well as a variety of 3' damages that arise upon oxidation or as products of enzymatic processing. If left unrepaired, APE1 substrates can promote mutagenic and cytotoxic outcomes. We describe herein a dominant-negative form of APE1 that lacks detectable nuclease activity and binds substrate DNA with a 13-fold higher affinity than the wild-type protein. This mutant form of APE1, termed ED, possesses two amino acid substitutions at active site residues Glu(96) (changed to Gln) and Asp(210) (changed to Asn). In vitro biochemical assays reveal that ED impedes wild-type APE1 AP site incision function, presumably by binding AP-DNA and blocking normal lesion processing. Moreover, tetracycline-regulated (tet-on) expression of ED in Chinese hamster ovary cells enhances the cytotoxic effects of the laboratory DNA-damaging agents, methyl methanesulfonate (MMS; 5.4-fold) and hydrogen peroxide (1.5-fold). This MMS-induced, ED-dependent cell killing coincides with a hyperaccumulation of AP sites, implying that excessive DNA damage is the cause of cell death. Because an objective of the study was to identify a protein reagent that could be used in targeted gene therapy protocols, the effects of ED on cellular sensitivity to a number of chemotherapeutic compounds was tested. We show herein that ED expression sensitizes Chinese hamster ovary cells to the killing effects of the alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (also known as carmustine) and the chain terminating nucleoside analogue dideoxycytidine (also known as zalcitabine), but not to the radiomimetic bleomycin, the nucleoside analogue beta-D-arabinofuranosylcytosine (also known as cytarabine), the topoisomerase inhibitors camptothecin and etoposide, or the cross-linking agents mitomycin C and cisplatin. Transient expression of ED in the human cancer cell line NCI-H1299 enhanced cellular sensitivity to MMS, 1,3-bis(2-chloroethyl)-1-nitrosourea, and dideoxycytidine, demonstrating the potential usefulness of this strategy in the treatment of human tumors.
Highlights
Genetic damage can arise via a variety of means, most notably via spontaneous decay, reactions with intracellular chemicals, aberrant enzymatic processing events, and modification by environmental or clinical DNA-damaging agents [1,2,3,4]
We postulated that simultaneous mutation of these two amino acids would create a catalytically inactive AP endonuclease 1 (APE1) protein with improved DNA-binding capacity
Using a previously established electrophoretic mobility shift assay, we found that ED displayed an f13-fold higher affinity for AP-DNA compared with wild-type APE1 protein (Fig. 1D); this enhanced affinity is presumably due to the loss of the repulsion between the negatively charged acidic residues and the phosphate backbone of DNA
Summary
Genetic damage can arise via a variety of means, most notably via spontaneous decay, reactions with intracellular chemicals, aberrant enzymatic processing events, and modification by environmental or clinical DNA-damaging agents [1,2,3,4]. The most common intermediates or forms of DNA damage include base modifications, abasic sites, and singlestrand breaks. These lesions are typically recognized and removed by the concerted effort of proteins that function in the base excision repair (BER) pathway [5]. More complex lesions, such as DNA double-strand breaks, are corrected by recombinational repair responses [6]. Defects in participants of BER have been associated with increased sensitivity to DNA-damaging agents, genomic instability, cancer susceptibility, neurodegeneration, premature aging phenotypes, and other human diseases [7]
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