A murine AP-endonuclease gene-targeted deficiency with post-implantation embryonic progression and ionizing radiation sensitivity

Abstract

Apurinic/apyrimidinic endonuclease (here designated APE/REF) carries out repair incision at abasic or single-strand break damages in mammals. This multifunctional protein also has putative role(s) as a cysteine `reducing factor' (REF) in cell-stress transcriptional responses. To assess the significance of APE/REF for embryonic teratogenesis we constructed a more precisely targeted Ape/Ref-deficient genotype in mice. Ape/Ref gene replacement in ES cells eliminated the potential of APE/REF protein synthesis while retaining the Ape/Ref bi-directional promoter that avoided potential inactivation of an upstream gene. Chimeric animals crossed into Tac:N:NIHS-BC produced germline transmission. Homozygous null Ape/Ref-embryos exhibited successful implantation and nearly normal developmental progression until embryonic day 7.5 followed by morphogenetic failure and adsorption of embryos by day 9.5. We characterized the cellular events proceeding to embryonic lethality and examined ionizing radiation sensitivity of pre-implantation Ape/Ref-null embryos. After intermating of heterozygotes, Mendelian numbers of putative Ape/Ref-null progeny embryos at day 6.5 displayed a several-fold elevation of pycnotic, fragmenting cell nuclei within the embryo proper—the epiblast. Increased cell-nucleus degeneration occurred within epiblast cells while mitosis continued and before obvious morphogenetic disruption. Mitogenic response to epiblast cell death, if any, was ineffective for replacement of lost cells. Extra-embryonic yolk sac, a trophectoderm derived lineage retained normal appearance to day 9. Explanted homozygous Ape/Ref-null blastocysts displayed increased sensitivity to γ-irradiation, most likely a manifestation of APE/REF incision defect. Our study establishes that this new Ape/Ref deficiency genotype is definitely capable of post-implantation developmental progression to the onset of gastrulation. Function(s) of APE/REF in base damage incision and also conceivably in mitogenic responses towards epiblast cell death are critical for transit through the gastrulation stage of embryonic growth and development.

Introduction

Endogenous DNA damages have poorly understood developmental and physiological consequences. Among spontaneous damages, DNA apurinic/apyrimidinic (AP) sites arise by hydrolytic scission of nucleotide N-glycosyl bonds (for reviews, see Refs. 1, 2). AP-site(s) also comprise intermediates in the repair of several kinds of DNA base modifications. Indirect evidence of excreted urinary metabolites gave estimates of 10⁴ to 10⁵ total excised base damages per cell per day. Base damages result from reactive oxygen species generated as byproducts of normal oxidative metabolism, as well as processes of spontaneous methylation, deamination and depurination 3, 4, 5. Sensitive measurement of AP-sites using an AP-site aldehyde reactive probe is consistent with these estimates 6, 7. AP-sites also comprise a predominant family of lesions generated by cell exposure to γ-ionizing radiation 1, 8, 9. Removal of AP-sites and certain single strand break damages occurs by a ubiquitous pathway of base excision repair. Indeed, investigators have purified all of the components necessary and sufficient for base excision repair in vitro 10, 11. Briefly, specific N-glycosylases release the altered base producing an AP-site intermediate. Apurinic/apyrimidinic endonuclease (APE/REF) then catalyses phosphodiester backbone cleavage immediately 5′ to the AP-site resulting in an available 3′-OH. DNA polymerase-β then excises the 5′ deoxyribose phosphate (5′ dRP) as the ring-open aldehyde and polymerizes a single base. DNA ligase III with participation of the XRCC1 protein (a stabilizing factor [12]) act together to ligate the `short' patch. A `long patch' variant of base repair occurs following APE/REF-incision, if DNA polymerase-β cannot excise altered forms of AP-phosphoribose, for example a 3′ dRP or altered 5′ dRPs 13, 14, 15, 16, 17. Long patch base repair utilizes proliferating cell nuclear antigen (PCNA) and flap endonuclease (FEN-1/DNAase IV) to remove a short displaced two- to seven-base oligonucleotide followed by resynthesis.

A central component of base repair is therefore APE/REF multifunctional enzyme designated variously as APE, HAP-1, APEX, or Ref-1 18, 19, 20, 21. In addition to its endonuclease activity APE/REF can carry out 3′-diesterase, RNaseH and 3′–5′ exonuclease reactions (reviewed in Ref. [25]). These catalytic activities suggest that APE/REF may participate in removal of blocked 3′ or 5′ moieties from single strand breaks and from sites incised by 3′ AP-lyase N-glycosylases [2]. The conservation of the basic endonuclease enzymatic structure from E. coli exonuclease III to mammalian APE/REF emphasizes its importance in participating broadly in the cell defense for major classes of premutagenic lesions (reviewed in Ref. [26]). An alternate function (REF) is its cysteine oxido-reductase activity for various transcription factors 21, 22. The N-terminal region of APE/REF carries the REF function, cys65 (in human APE/REF) comprises the redox active center and cys93 is a regulatory cysteine [22]. Redox activity of APE/REF is regulated by interaction with thioredoxin [23]. Redox activity of APE/REF may therefore participate in cellular stress transcriptional responses and mitogenic signal transduction 23, 24. The role of REF function for normal development is unknown.

With high incidence of intrinsic AP-sites 3, 7the lack of repair incision for base and certain SSB damages may cause AP-site induced mutagenesis, DNA breakage and compromise embryonic development. Previous gene-targeted deficiencies of key base repair related genes, Ape/Ref [27]and Polβ [28]exhibited onsets of murine embryo lethality ranging from peri-implantation (day 4–5; Ape/Ref) until mid-gestation (day 10+) for Polβ homozygotes. The difference in timing of morphogenetic failures suggests that base repair deficiency may not be the only explanation of embryo lethality. We re-examined aspects of this question with a precise Ape/Ref⁻ gene-targeted disruption bred into the 129:NIHS hybrid genetic background. Progeny of Ape/Ref⁻ heterozygote intermating first showed spatially localized degeneration of epiblast cell nuclei at embryonic day 6.5. At this time the embryos were still growing. Embryo morphogenetic failure started at gastrulation—day 7.5. Ape/Ref^−/− blastocysts also displayed increased γ-radiosensitivity that arose most likely by loss of incision repair function of APE/REF. These phenotypes suggest that intrinsic teratogenic events consistent with DNA damages in Ape/Ref deficient embryos lead to excessive epiblast cell death and ineffective mitogenic response. We discuss these phenotypes in terms of multifunctional roles of APE/REF.