Inactivation of a common OGG1 variant by TNF-alpha in mammalian cells.

Reactive oxygen species threaten genomic integrity by inducing oxidative DNA damage. One common form of oxidative DNA damage is the mutagenic lesion 8-oxoguanine (8-oxodG). One driver of oxidative stress that can induce 8-oxodG is inflammation, which can be initiated by the cytokine tumor necrosis factor alpha (TNF-α). Oxidative DNA damage is primarily repaired by the base excision repair pathway, initiated by glycosylases targeting specific DNA lesions. 8-oxodG is excised by 8-oxoguanine glycosylase 1 (OGG1). A common Ogg1 allelic variant is S326C-Ogg1, prevalent in Asian and Caucasian populations. S326C-Ogg1 is associated with various forms of cancer, and is inactivated by oxidation. However, whether oxidative stress caused by inflammatory cytokines compromises OGG1 variant repair activity remains unknown. We addressed whether TNF-α causes oxidative stress that both induces DNA damage and inactivates S326C-OGG1 via cysteine 326 oxidation. In mouse embryonic fibroblasts, we found that S326C-OGG1 was inactivated only after exposure to H2O2 or TNF-α. Treatment with the antioxidant N-acetylcysteine prior to oxidative stress rescued S326C-OGG1 activity, demonstrated by in vitro and cellular repair assays. In contrast, S326C-OGG1 activity was unaffected by potassium bromate, which induces oxidative DNA damage without causing oxidative stress, and presumably cysteine oxidation. This study reveals that Cys326 is vulnerable to oxidation that inactivates S326C-OGG1. Physiologically relevant levels of TNF-α simultaneously induce 8-oxodG and inactivate S326C-OGG1. These results suggest a mechanism that could contribute to increased risk of cancer among S326C-Ogg1 homozygous individuals.

Repair efficiency of (5'S)-8,5'-cyclo-2'-deoxyguanosine and (5'S)-8,5'-cyclo-2'-deoxyadenosine depends on the complementary base.

5'-R and 5'-S diastereoisomers of 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG) containing a base-sugar covalent bond are formed by hydroxyl radicals. R-cdA and S-cdA are repaired by nucleotide excision repair (NER) in mammalian cellular extracts. Here, we have examined seven purified base excision repair enzymes for their ability to repair S-cdG or S-cdA. We could not detect either excision or binding of these enzymes on duplex oligonucleotide substrates containing these lesions. However, both lesions were repaired by HeLa cell extracts. Dual incisions by human NER on a 136-mer duplex generated 24-32 bp fragments. The time course of dual incisions were measured in comparison to cis-anti-B[a]P-N(2)-dG, an excellent substrate for human NER, which showed that cis-anti-B[a]P-N(2)-dG was repaired more efficiently than S-cdG, which, in turn, was repaired more efficiently than S-cdA. When NER efficiency of S-cdG with different complementary bases was investigated, the wobble pair S-cdG·dT was excised more efficiently than the S-cdG·dC pair that maintains nearly normal Watson-Crick base pairing. But S-cdG·dA mispair with no hydrogen bonds was excised less efficiently than the S-cdG·dC pair. Similar pattern was noted for S-cdA. The S-cdA·dC mispair was excised much more efficiently than the S-cdA·dT pair, whereas the S-cdA·dA pair was excised less efficiently. This result adds to complexity of human NER, which discriminates the damaged base pairs on the basis of multiple criteria.

Saccharomyces cerevisiae Apn1 mutation affecting stable protein expression mimics catalytic activity impairment: implications for assessing DNA repair capacity in humans.

Apurinic/apyrimidinic (AP) endonucleases play a major role in the repair of AP sites, oxidative damage and alkylation damage in DNA. We employed Saccharomyces cerevisiae in an unbiased forward genetic screen to identify amino acid substitutions in the major yeast AP endonuclease, Apn1, that impair cellular DNA repair capacity by conferring sensitivity to the DNA alkylating agent methyl methanesulfonate. We report here the identification and characterization of the Apn1 V156E amino acid substitution mutant through biochemical and functional analysis. We found that steady state levels of Apn1 V156E were substantially decreased compared to wild type protein, and that this decrease was due to more rapid degradation of mutant protein compared to wild type. Based on homology to E. coli endonuclease IV and computational modeling, we predicted that V156E impairs catalytic ability. However, overexpression of mutant protein restored DNA repair activity in vitro and in vivo. Thus, the V156E substitution decreases DNA repair capacity by an unanticipated mechanism via increased degradation of mutant protein, leading to substantially reduced cellular levels. Our study provides evidence that the V156 residue plays a critical role in Apn1 structural integrity, but is not involved in catalytic activity. These results have important implications for elucidating structure-function relationships for the endonuclease IV family of proteins, and for employing simple eukaryotic model systems to understand how structural defects in the major human AP endonuclease APE1 may contribute to disease etiology.

Cigarette smoke induces DNA damage and alters base-excision repair and tau levels in the brain of neonatal mice.

The prenatal and perinatal periods of brain development are especially vulnerable to insults by environmental agents. Early life exposure to cigarette smoke (CS), which contains both genotoxicants and oxidants, is considered an important risk factor for both neurodevelopmental and neurodegenerative disorders. Yet, little is known regarding the underlying pathogenetic mechanisms. In the present study, neonatal Swiss ICR (CD-1) albino mice were exposed to various concentrations of CS for 4 weeks and the brain examined for lipid peroxides, DNA damage, base-excision repair (BER) enzymes, apoptosis, and levels of the microtubule protein tau. CS induced a dose-dependent increase in both malondialdehyde and various types of DNA damage, including single-strand breaks, double-strand breaks, and DNA-protein cross-links. However, the CS-induced DNA damage in the brain returned to basal levels 1 week after smoking cessation. CS also modulated the activity and distribution of the BER enzymes 8-oxoguanine-DNA-glycosylase (OGG1) and apyrimidinic/apurinic endonuclease (APE1) in several brain regions. Normal tau (i.e., three-repeat tau, 3R tau) and various pathological forms of tau were also measured in the brain of CS-exposed neonatal mice, but only 3R tau and tau phosphorylated at serine 199 were significantly elevated. The oxidative stress, genomic dysregulation, and alterations in tau metabolism caused by CS during a critical period of brain development could explain why CS is an important risk factor for both neurodevelopmental and neurodegenerative disorders appearing in later life.

Characterization of a multifunctional PEG-based gene delivery system containing nuclear localization signals and endosomal escape peptides.

Endosomal escape and nuclear localization are two barriers to gene delivery that need to be addressed in the design of new nonviral gene delivery vehicles. We have previously synthesized low-toxicity polyethylene glycol (PEG)-based vehicles with endosomal escape functionalities, but it was determined that the transfection efficiency of PEG-based vehicles that escaped the endosome was still limited by poor nuclear localization. Two different nuclear localization signal (NLS) peptides, SV40 and TAT, were coupled to PEG-based vehicles with DNA-binding peptides (DBPs) to determine the effect of NLS peptides on the transfection efficiency of PEG-based gene delivery vehicles. Coupling one SV40 peptide, a classical NLS, or two TAT peptides, a nonclassical NLS, to PEG-DBP vehicles increased the transfection efficiency of PEG-DBP/DNA particles 15-fold and resulted in similar efficiency to that of a common cationic polymer vehicle, polyethylenimine (PEI). The transfection efficiency of both types of PEG-DBP-NLS particles was further increased 7-fold in the presence of chloroquine, suggesting that the transfection efficiency of PEG-DBP-NLS particles is limited by their ability to escape the endosome. To develop particles that could escape the endosome and target the nucleus, a mixture of PEG-DBP-NLS vehicles and PEG-based vehicles with DBPs and endosomal escape peptides were complexed with plasmid DNA to form multifunctional particles that had a transfection efficiency 2-3 times higher than that of PEI. Additionally, the PEG-based vehicles were less toxic and more resistant to nonspecific protein adsorption than PEI, making them an attractive alternative for nonviral gene delivery.

The effect of endosomal escape peptides on in vitro gene delivery of polyethylene glycol-based vehicles.

BACKGROUND: With recent progress in gene therapy clinical trials, there is an even greater demand to advance the development of nonviral gene delivery vehicles. We have previously developed poly(ethylene glycol) (PEG)-based vehicles with transfection efficiency similar to polyethyleneimine and low cytotoxicity. It was hypothesized that conjugating endosomal escape peptides (EEPs) to PEG-based vehicles would further increase their transfection efficiency. The present study aimed to determine how two different EEPs, INF7 and H5WYG, which destabilize the endosomal membrane at different pHs, affect the efficiency of PEG-based vehicles. METHODS: INF7 and H5WYG were conjugated to PEG-tetraacrylate (PEG-TA) via a Michael-type addition at the desired molar ratios. The pH-dependent membrane lytic activity, transfection efficiency, particle size, zeta potential, and endosomal escape kinetic rate constants were determined. RESULTS: Fusogenic peptides, INF7 and H5WYG, showed pH-dependent membrane lytic activity when conjugated to PEG-TA. The highest membrane lytic activity of PEG-INF7 and PEG-H5WYG conjugates occurred at pH 5 and 5.5, respectively. Coupling one INF7 peptide to PEG-DNA binding peptide (DBP) vehicles increased the transfection efficiency ten-fold and showed greater transfection efficiency than PEG-DBP vehicles coupled with H5WYG peptide. Fitting a first-order kinetic model to the average intracellular pH of the vehicle/DNA particles over time determined that coupling EEPs to PEG-DBP vehicles increased the endosomal escape rate constant by two orders of magnitude. CONCLUSIONS: Endosomal escape is a key step in nonviral cellular trafficking and thus the transfection efficiency of nonviral vehicles can be increased by targeting release of DNA from the endosome with EEPs.