Mutation Research/DNA Repair
A murine AP-endonuclease gene-targeted deficiency with post-implantation embryonic progression and ionizing radiation sensitivity
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.
Section snippets
Targeting vector
The targeting vector (pAPE-neo-TK) derives from an 11 kb BglII subclone from a cosmid of 129Sv/J strain mouse genomic DNA [29](Fig. 1). It contains a total of 10 kb of homology (1.2 kb short arm and 8.8 kb long arm) with the Ape/Ref locus. The construct contains a deletion of 675 bp from a Bsu36I site 20 bp upstream of, and including the ribosome binding site and translation start ATG for Ape/Ref. This left the next inframe ATG at amino acid 268 producing complete translational disruption. The
Targeted disruption of the Ape/Ref gene in mouse ES cells
The Ape/Ref neo-TK targeting construct replaced an allele of Ape/Ref in 61 of 212 embryonic stem (ES) cell clones (29%). The high efficiency of homologous gene replacement selection was undoubtedly due to the extent of homologous sequences in the targeting construct (10 kb); the use of isogenic DNA (129Sv/J); and double drug selection [32]. Two normal karyotype cell clones were injected into C57BL/6J host blastocysts. Both ES clones (ES10, ES42) produced chimeric mice outcrossed to
A new genetic construction for Ape/Ref-deficiency
The Ape/Ref gene encoding the major nucleus apurinic/apyrimidinic endonuclease in mammals has been subject of extensive biochemical and structural analyses in vitro, while its biological functions are much less well characterized. A generalized deficiency of base excision repair would increase rates of spontaneous mutation and accumulation of DNA breaks 38, 39, 40, 41. These events might be the cause of peri-implantation inviability, as has been previously suggested for APE/REF deficient
Acknowledgements
This work was supported by funding from the US Department of Energy to D.J.C. and a grant from the NIH to R.A.P. M.A.M. was supported in part by a LANL-UC Directed Research and Development Visiting Scholar Grant while visiting UCSF.
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- 1
Current address: ImClone Systems, 180 Varick Street, New York, NY 10014.
- 2
DLL and MAM contributed equally to the work.