This work is proposed to investigate the major mammalian apurinic-apyrimidinic endonuclease coordination of its DNA oxidative damage base repair function with its roles in regulation of gene transcription. AP-sites in large numbers form spontaneously or as intermediates in glycosylase-initiated base repair. Repair of AP-sites is imperative both for transcription maintenance and to prevent accumulation of base substitution mutations and strand breaks which may lead to chromosome aberrations. The intriguing bifunctional linkage of a key AP endonuclease (termed APE, HAP1, APEX) with an oxidation-reduction (redox) transcription factor activities (termed Redox Factor-1' or REF-1) motivates our alternative working hypotheses: APE/REF protein is cell nucleus-localized by interaction with transcription factors which may effect an improvement of base repair efficiency, or alternatively REF function facilitates auto-induction adaptive responses to low level genotoxins or cell stresses to increase cellular base repair capacity. These hypotheses may not be mutually exclusive. We have produced a new, more precise APE/REF gene-targeted deletion in mice, and are complementing its lethality by APE/REF transgenes and by gene-targeted replacement of the wildtype locus by mutant REF or APE functional alleles. We will test three specific hypotheses: 1. whether transgene or gene-targeted mutations of redox-function (REF) in particular modulate in vivo DNA repair competency and cell stress response to hypoxia. This hypothesis will be tested by assessing the level of REF-mutant transgene or mutant allele complementation of a CRE-LOX induced null allele in cell cultures exposed to sub-toxic or cytotoxic doses of ionizing radiation, drugs, pro-oxidant chemicals and hypoxia. 2. We will determine whether REF mutant alleles and transgenes can carry out the transcriptional regulatory interactions of APE/REF oxidoreductase in cells starting with AP-1 (cJun/cFos). 3. We will test the hypothesis that transgenes and mutant REF alleles will demonstrate an inverse correlation between cell base repair capacity, in vivo and levels of chemical- and radiation-induced AP-sites and sister chromatid exchanges. This three (3) year research plan will elucidate the extent of putative coupling between nucleus localization, transcriptional oxidoreductase function and DNA repair biological role(s) of APE/REF in vivo for amelioration of base damages and oxidative cell stress. Future directions include: A) examination APE/REF interactions for low-dose thresholds of redox cell activation in response to chronic or sub-acute cell stresses and to assess the relevance of APE/REF mutations for amyotrophic lateral sclerosis (Lou Gehrig s).
NIH ExPorter
NIH ExPorter