Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
Rapid communicationEvolution of the redox function in mammalian apurinic/apyrimidinic endonuclease
Introduction
Redox regulation has been shown to play an important role in modulating the DNA binding activity of a number of transcription factors including AP-1, NFκB, HIF-1α, HLF, CREB, PAX, p53 and others [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11] (reviewed in [12]). As described for AP-1 (Fos/Jun), this regulation involves the reduction of a critical Cys residue(s) within c-Jun that allows it to bind DNA with much higher affinity than the oxidized c-Jun [13], [14]. The factor responsible for reducing AP-1 was found to be a nuclear factor, termed redox effector factor 1 (Ref1) [15], and later identified as apurinic/apyrimidinic endonuclease (Ape1, also referred to as HAP1 or APEX1) [2]. Ape1 is an essential enzyme in the base excision repair pathway catalyzing the second step in the pathway, cleavage of the phosphodiester backbone 5′ of the abasic site leaving a 3′ hydroxyl and 5′-deoxyribose phosphate. Deletion of the APE1 gene results in very early embryonic lethality in mice [16], and knock-down of Ape through the use of a morpholino oligonucleotide injected in zebrafish embryos results in death at the midblastula transition [17]. In addition, Ape has been shown to be involved in heart and blood development in zebrafish [17].
The redox activity has been shown to require the N-terminal region of the protein (reviewed in [12]). The human Ape1 includes 61 residues not present in exonuclease III from E. coli, and while truncations of up to 40 residues do not affect the redox activity, removal of the 62 N-terminal residues from the human Ape1 renders this enzyme redox inactive [14], [18]. It was hypothesized that Ape1's redox activity might involve a Cys residue [14]. Accordingly, each of the seven Cys residues in Ape1 was substituted individually with Ala, and the resulting enzymes were tested for their ability to reduce AP-1 thereby allowing it to bind target DNA specifically in an electrophoretic mobility shift assay (EMSA) [18]. Of these single Cys mutants, all when reduced retained wild-type redox activity except C65A hApe1, which was redox inactive. When oxidized, C93A hApe1 was found to be redox active, while all of the other mutants were inactive. Thus, a redox mechanism involving a disulfide bond between Cys 65 and Cys 93 was proposed [18].
Subsequently, however, several crystal structures of Ape1 have been reported [19], [20], [21] providing three important observations with regard to the redox activity of Ape1. First, no disulfide bonds are present in any of the structures. Second, Cys 65 is a buried residue in the conformation of hApe1 observed in the crystal structures and therefore not accessible to the transcription factors that Ape1 reduces. Third, the structures of hApe1 that have been reported to date in different crystallographic lattices are remarkably similar. Even in the hApe1–DNA complex, there are no significant conformational changes in the enzyme [20]. Thus, the mechanism by which Cys 65 would participate in a potential redox reaction between Ape1 and a transcription factor requires further investigation. One possible explanation offered by Gorman et al. [19] was that substitution of Cys 65 with Ala may affect the stability and/or folding of Ape1 and result in subsequent loss of the redox activity. More recently, the role of Cys 65 in Ape1's redox activity has been challenged through the creation of a Ref1C64A/C64A mouse (C64 is equivalent to the human C65 residue) that was shown to develop normally [22]; in this study the authors concluded that Cys 65 is not required for redox activity.
Ape vertebrate sequences are highly conserved with the most distantly related sequence, that of zebrafish Ape (zApe) being 66% identical to the human Ape1. We had initially selected zebrafish as a potential model system to study the role of Ape in development as mice knockouts were lethal. However, as reported here, the wild-type zApe lacks redox activity. Thus, we have not pursued the zebrafish development experiments. We report the results of biochemical studies including hApe1, zApe, and a zApe mutant engineered to acquire redox function, the first crystal structures of redox inactive enzymes from vertebrates, and a discussion of the mechanistic implications for the redox activity of Ape.
Section snippets
Evolutionary analysis of Ape sequences
Orthologous sequences for APE1 in human and other species except zebrafish were obtained from the 28 species alignment at the UCSC genome browser [23]. To obtain the zebrafish APE1 gene sequence, the BLAT web server at UCSC was used to search for matches to the human APE1 gene within the zebrafish genome. We next identified the 3-nucleotide codons corresponding to the 7 human Cys residues in the APE1 genes in all species and aligned the concatenated Cys codons using CLUSTALW [24]. The branch
Cell-based analysis of redox activity for hApe1 Cys mutants
The human Ape1 includes seven Cys residues, 65, 93, 99, 138, 208, 296, and 310. In a previous study [18], the redox activity of single Cys mutants was assessed using an in vitro EMSA redox assay in which reduction of AP-1 by hApe1 results in a shifted band, indicating the formation of an AP-1/DNA complex. Of the single Cys mutants tested, only C65A hApe1 was found to be redox inactive [18].
In order to assess the redox activity of Ape1 and single Cys mutants within a cell providing data
Discussion
The results presented here impact our current understanding of the mechanism of Ape1's redox activity. First, our evolutionary sequence analysis suggests that the redox function is found only in mammals and results from the acquisition of Cys 65. As shown in Table 2, the equivalent residue is either Ser or Thr in non-mammalian vertebrate Ape sequences. While Cys 138 is also conserved in most mammals and not in non-mammalian vertebrates, the platypus has an Arg residue in the equivalent
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
We thank Steve Ginell, Marianne Cuff and Andrzej Joachimiak from the Structural Biology Center Collaborative Access Team at the Advanced Photon Source (APS), Jay Nix from the Molecular Biology Consortium at the Advanced Light Source (ALS), members of the Georgiadis and Kelley laboratories, and Tom Hurley for helpful discussions. Data were collected at beamline 19-ID in the facilities of the SBC-CAT at APS and at beamline 4.2.2 at the ALS. Results shown in this report are derived from work
References (39)
- et al.
A redox mechanism controls differential DNA binding activities of hypoxia-inducible factor (HIF) 1alpha and the HIF-like factor
J. Biol. Chem.
(2000) - et al.
Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit
J. Biol. Chem.
(1996) - et al.
Redox regulation of the DNA binding activity in transcription factor PEBP2. The roles of two conserved cysteine residues
J. Biol. Chem.
(1997) - et al.
Going APE over ref-1
Mutat. Res.
(2000) - et al.
Two divalent metal ions in the active site of a new crystal form of human apurinic/apyrimidinic endonuclease, Ape1: implication for the catalytic mechanism
J. Mol. Biol.
(2001) - et al.
Processing of X-ray diffraction data collected in oscillation mode
Methods Enzymol.
(1997) - et al.
Raster3d: photorealistic molecular graphics
Methods Enzymol.
(1997) - et al.
Identification and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA binding activity
EMBO J.
(1992) - et al.
Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme
EMBO J.
(1992) - et al.
Activation of AP-1 and of a nuclear redox factor, Ref-1, in the response of HT29 colon cancer cells to hypoxia
Mol. Cell. Biol.
(1994)
Molecular mechanisms of transcription activation by HLF and HIF1alpha in response to hypoxia: their stabilization and redox signal-induced interaction with CBP/p300
EMBO J.
Identification of redox/repair protein Ref-1 as a potent activator of p53
Genes Dev.
Redox factor 1 (Ref-1) enhances specific DNA binding of p53 by promoting p53 tetramerization
Oncogene
Ref-1 regulates the transactivation and pro-apoptotic functions of p53 in vivo
EMBO J.
Characterization of the DNA-binding properties of the early growth response-1 (Egr-1) transcription factor: evidence for modulation by a redox mechanism
DNA Cell Biol.
Redox regulation of Fos and Jun DNA-binding activity in vitro
Science
The redox and DNA-repair activities of Ref-1 are encoded by nonoverlapping domains
Proc. Natl. Acad. Sci. U.S.A.
A ubiquitous nuclear protein stimulates the DNA-binding activity of fos and jun indirectly
Cell Growth Differ.
The redox/DNA repair protein, Ref-1, is essential for early embryonic development in mice
Proc. Natl. Acad. Sci. U.S.A.
Cited by (108)
Apurinic/apyrimidinic endonuclease 1 regulates palmitic acid-mediated apoptosis in cardiomyocytes via endoplasmic reticulum stress
2023, Biochemical and Biophysical Research CommunicationsPre-steady-state kinetic and mutational insights into mechanisms of endo- and exonuclease DNA processing by mutant forms of human AP endonuclease
2022, Biochimica et Biophysica Acta - General SubjectsThioredoxin reductase as a pharmacological target
2021, Pharmacological Research