Citation: Xue, Z.; Demple, B. Knockout and Inhibition of Ape1: Roles of Ape1 in Base Excision DNA Repair and Modulation of Gene Expression. Antioxidants 2022, 11, 1817. https://doi.org/10.3390/ antiox11091817 Academic Editors: Marina Roginskaya, Yuriy Razskazovskiy and Alexandros Georgakilas Received: 21 July 2022 Accepted: 7 September 2022 Published: 15 September 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). antioxidants Article Knockout and Inhibition of Ape1: Roles of Ape1 in Base Excision DNA Repair and Modulation of Gene Expression Zhouyiyuan Xue 1,2 and Bruce Demple 1, * 1 Department of Pharmacological Sciences, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY 11794-8651, USA 2 Molecular and Cellular Biochemistry Program, Stony Brook University, Stony Brook, NY 11794-8651, USA * Correspondence: bruce.demple@stonybrook.edu; Tel.: +1-(631)-444-3978 Abstract: Apurinic/apyrimidinic endonuclease 1/redox effector-1 (Ape1/Ref-1) is the major apurin- ic/apyrimidinic (AP) endonuclease in mammalian cells. It functions mainly in the base excision repair pathway to create a suitable substrate for DNA polymerases. Human Ape1 protein can activate some transcription factors to varying degrees, dependent on its N-terminal, unstructured domain, and some of the cysteines within it, apparently via a redox mechanism in some cases. Many cancer studies also suggest that Ape1 has potential for prognosis in terms of the protein level or intracellular localization. While homozygous disruption of the Ape1 structural gene APEX1 in mice causes embryonic lethality, and most studies in cell culture indicate that the expression of Ape1 is essential, some recent studies reported the isolation of viable APEX1 knockout cells with only mild phenotypes. It has not been established by what mechanism the Ape1-null cell lines cope with the endogenous DNA damage that the enzyme normally handles. We review the enzymatic and other activities of Ape1 and the recent studies of the properties of the APEX1 knockout lines. The APEX1 deletions in CH12F3 and HEK293 FT provide an opportunity to test for possible off-target effects of Ape1 inhibition. For this work, we tested the Ape1 endonuclease inhibitor Compound 3 and the redox inhibitor APX2009. Our results confirmed that both APEX1 knockout cell lines are modestly more sensitive to killing by an alkylating agent than their Ape1-proficient cells. Surprisingly, the knockout lines showed equal sensitivity to direct killing by either inhibitor, despite the lack of the target protein. Moreover, the CH12F3 APEX1 knockout was even more sensitive to Compound 3 than its APEX1 + counterpart. Thus, it appears that both Compound 3 and APX2009 have off-target effects. In cases where this issue may be important, it is advisable that more specific endpoints than cell survival be tested for establishing mechanism. Keywords: Ape1; APEX1; base excision repair; redox; Ape1 inhibitor; knockout; CH12F3; HEK293 FT; Compound 3; APX2009 1. Introduction Genomic DNA is under the relentless threat of molecular decay due to endogenous and exogenous damage, which threatens genetic stability. Reactive oxygen species (ROS), notably the byproducts of aerobic metabolism, produce an array of DNA lesions, including 8-oxoguanine (8-oxoG) and thymine glycol. Hydrolysis and various other reactive metabo- lites add many other DNA lesions to this burden [1]. Many of these lesions disrupt DNA structure only slightly or not at all, but they nonetheless can exert mutagenic or cytotoxic effects if left unrepaired. Ring-opened aldehyde apurinic/apyrimidinic (AP) sites can form a covalent bond with neighboring nucleotides or histone lysines, which can result in highly toxic DNA interstrand cross-links and histone-DNA cross-links [2,3]. Base excision DNA repair (BER) is the frontline process that deals with most of the small lesions. Following the removal of a damaged base by a DNA glycosylase, the 5 ′ - phosphodiester bond of the resulting apurinic/apyrimidinic (AP) site is cleaved by Ape1; Antioxidants 2022, 11, 1817. https://doi.org/10.3390/antiox11091817 https://www.mdpi.com/journal/antioxidants