Inactivation by oxidation and recruitment into stress granules of hOGG1 but not APE1 in human cells exposed to sub-lethal concentrations of cadmium.

The induction of mutations in mammalian cells exposed to cadmium has been associated with the oxidative stress triggered by the metal. There is increasing evidence that the mutagenic potential of Cd is not restricted to the induction of DNA lesions. Cd has been shown to inactivate several DNA repair enzymes. Here we show that exposure of human cells to sub-lethal concentrations of Cd leads to a time- and concentration-dependent decrease in hOGG1 activity, the major DNA glycosylase activity responsible for the initiation of the base excision repair (BER) of 8-oxoguanine, an abundant and mutagenic form of oxidized guanine. Although there is a slight effect on the level of hOGG1 transcripts, we show that the inhibition of the 8-oxoguanine DNA glycosylase activity is mainly associated with an oxidation of the hOGG1 protein and its disappearance from the soluble fraction of total cell extracts. Confocal microscopy analyses show that in cells exposed to Cd hOGG1-GFP is recruited to discrete structures in the cytoplasm. These structures were identified as stress granules. Removal of Cd from the medium allows the recovery of the DNA glycosylase activity and the presence of hOGG1 in a soluble form. In contrast to hOGG1, we show here that exposure to Cd does not affect the activity of the second enzyme of the pathway, the major AP endonuclease APE1.

Expression of 8-oxoguanine DNA glycosylase (Ogg1) in mouse retina.

PURPOSE: The retina is highly exposed to oxidative stress due to the high level of oxygen consumption in this tissue and its exposure to light. The main DNA base lesion generated by oxygen free radicals is 8-oxoguanine (8-oxoG). However, its presence in retinal cells and the mechanisms underlying its repair remain undetermined. METHODS: 8-oxoguanine DNA glycosylase (Ogg1) gene expression and messenger localization in adult mouse ocular tissues was analyzed by RT-PCR and in situ hybridization. Using immunohistochemistry, we determined the localization of Ogg1 protein and three base excision repair (BER) enzymes: apurinic/apyrimidic endonuclease (APE1), DNA polymerase beta, and X-ray repair cross-complementation group 1 (XRCC1). Ogg1 and AP-lyase activities in the neuroretina were obtained using double-stranded oligonucleotides harboring either an 8-oxoG residue or a tetrahydrofuran. RESULTS: We report here that 8-oxoG is abundant in the retina. Ogg1, the enzyme responsible for the recognition and excision of the oxidized base, is present in its active form and found mainly in ganglion cells and photoreceptor inner segments. We show that APE1 and DNA polymerase beta, two BER proteins involved in 8-oxoG repair, are also present in these cells. The cellular distribution of these proteins was similar to that of Ogg1. XRRC1 is present in both inner nuclear and ganglion cells layers; however, this protein is absent from photoreceptor inner segments. CONCLUSIONS: This is the first study to demonstrate the presence of a functional 8-oxoG BER pathway in retinal neurons. The study of three BER proteins involved in 8-oxoG elimination demonstrates that XRCC1 localization differs from those of Ogg1, APE1, and DNA polymerase beta. This result suggests that the elimination of 8-oxoG is coordinated through two pathways, which differ slightly according to the cellular localization of the abnormally oxidized guanine.

Redox regulation of human OGG1 activity in response to cellular oxidative stress.

8-Oxoguanine (8-oxoG), a common and mutagenic form of oxidized guanine in DNA, is eliminated mainly through base excision repair. In human cells its repair is initiated by human OGG1 (hOGG1), an 8-oxoG DNA glycosylase. We investigated the effects of an acute cadmium exposure of human lymphoblastoid cells on the activity of hOGG1. We show that coinciding with alteration of the redox cellular status, the 8-oxoG DNA glycosylase activity of hOGG1 was nearly completely inhibited. However, the hOGG1 activity returned to normal levels once the redox cellular status was normalized. In vitro, the activity of purified hOGG1 was abolished by cadmium and could not be recovered by EDTA. In cells, however, the reversible inactivation of OGG1 activity by cadmium was strictly associated with reversible oxidation of the protein. Moreover, the 8-oxoG DNA glycosylase activity of purified OGG1 and that from crude extracts were modulated by cysteine-modifying agents. Oxidation of OGG1 by the thiol oxidant diamide led to inhibition of the activity and a protein migration pattern similar to that seen in cadmium-treated cells. These results suggest that cadmium inhibits hOGG1 activity mainly by indirect oxidation of critical cysteine residues and that excretion of the metal from the cells leads to normalization of the redox cell status and restoration of an active hOGG1. The results presented here unveil a novel redox-dependent mechanism for the regulation of OGG1 activity.

Oxidation status of human OGG1-S326C polymorphic variant determines cellular DNA repair capacity.

The hOGG1 gene encodes the DNA glycosylase that removes the mutagenic lesion 7,8-dihyro-8-oxoguanine (8-oxoG) from DNA. A frequently found polymorphism resulting in a serine to cysteine substitution at position 326 of the OGG1 protein has been associated in several molecular epidemiologic studies with cancer development. To investigate whether the variant allele encodes a protein with altered OGG1 function, we compared the 8-oxoG repair activity, both in vivo and in cell extracts, of lymphoblastoid cell lines established from individuals carrying either Ser/Ser or Cys/Cys genotypes. We show that cells homozygous for the Cys variant display increased genetic instability and reduced in vivo 8-oxoG repair rates. Consistently, their extracts have an almost 2-fold lower basal 8-oxoG DNA glycosylase activity when compared with the Ser variant. Treatment with reducing agents of either the Cys variant cells directly or of protein extracts from these cells increases the repair capacity to the level of the Ser variant, whereas it does not affect the activity in cells or extracts from the latter. Furthermore, the DNA glycosylase activity of cells carrying the Cys/Cys alleles is more sensitive to inactivation by oxidizing agents when compared with that of the Ser/Ser cells. Analysis of the redox status of the OGG1 protein in the cells confirms that the lower activity of OGG1-Cys326 is associated with the oxidation of Cys326 to form a disulfide bond. Our findings support the idea that individuals homozygous for the OGG1-Cys variant could more readily accumulate mutations under conditions of oxidative stress.