Cysteine 64 of Ref-1 Is Not Essential for Redox Regulation of AP-1 DNA Binding

The DNA binding activity of the AP-1 transcription factor complex is subject to reduction-oxidation (redox) regulation. This regulation involves posttranslational modification of a conserved cysteine residue in the DNA binding domains of Fos and Jun (2). The critical cysteine residue is highly conserved, and it is flanked by one or two basic amino acids in the four fos family members, the three jun family members, and at least four members of the ATF-CREB family of transcription factors. Chemical oxidation of the cysteines inhibits the DNA binding activity of Fos and Jun, and this inhibition is alleviated by reduction of the cysteines by thiol compounds or by a cellular protein, Ref-1 (43). Loss of redox regulation of AP-1 family members has biological effects. For example, in the oncogenic v-Jun protein, the cysteine has been replaced with a serine (26). Furthermore, serine substitution of the cysteine in Fos results in loss of redox regulation and enhancement of DNA binding and transforming potential (27). Ref-1 is also implicated in redox regulation of DNA binding of several other transcription factors, including NF-κB (43), Egr-1 (16), HIF-1α (7, 15, 25), HLF (7, 22), Pax-5 and Pax-8 (34-36), p53 (10, 18), and others. Ref-1 (also known as HAP1, Ape1, and APEX1) is a bifunctional enzyme. It was independently identified by its AP-1 redox activity (41, 42) and by its sequence identity to class II hydrolytic apurinic/apyrimidinic (AP) DNA repair endonucleases (4, 30, 31). These enzymes function in base excision repair of abasic DNA lesions generated by spontaneous hydrolysis or by exposure to reactive oxygen radicals (6). The DNA repair and redox regulatory activities of Ref-1 are carried out by physically separable domains of the protein (44). The dual activities of Ref-1 suggest that the repair of DNA lesions induced by reactive oxygen species and the regulation of the transcriptional response to an oxidizing cellular environment are tightly coupled. Immunodepletion studies using affinity-purified antibodies specific for Ref-1 demonstrated that Ref-1 is the major redox regulator of AP-1 DNA binding in HeLa cells (43). Furthermore, the essential role of Ref-1 in mammalian development was demonstrated by genetic inactivation of Ref-1 in mice (45). Embryos lacking a functional Ref-1 gene fail to develop beyond embryonic day 6. Death occurs following blastocyst formation, shortly after implantation. These embryos lack both the DNA repair and redox regulatory activities of Ref-1. Therefore, it is not possible to determine whether Ref-1 DNA repair activity, redox regulatory activity, or both are essential for viability. Ref-1 contains two cysteine residues within the redox-active domain (cysteines 64/65 and 93). Previous in vitro studies using recombinant human Ref-1 proteins suggested that cysteine 65 (cysteine 64 of mouse Ref-1) is the redox-active site of Ref-1 (39). These results suggested a sulfhydryl switch mechanism in which cysteine 64/65 of Ref-1 interacts with the conserved cysteines within the DNA binding domains of Fos and Jun and serves as a reductant. Here we report the first genetic analysis of the role of cysteine 64 in Ref-1 redox activity in vivo. Our findings demonstrate that Ref-1 cysteine 64 is not required for Ref-1 redox regulation of AP-1.