Rhoderick H. Elder

Endonuclease VIII-like (NEIL) 1 and 3 proteins eliminate oxidative DNA base damage and psoralen DNA interstrand crosslinks through initiation of base excision repair. Current evidence points to a DNA replication associated repair function of NEIL1 and NEIL3, correlating with induced expression of the proteins in S/G2 phases of the cell cycle. However previous attempts to express and purify recombinant human NEIL3 in an active form have been challenging. In this study, both human NEIL1 and NEIL3 have been expressed and purified from E. coli, and the DNA glycosylase activity of these two proteins confirmed using single- and double-stranded DNA oligonucleotide substrates containing the oxidative bases, 5-hydroxyuracil, 8-oxoguanine and thymine glycol. To determine the biochemical role that NEIL1 and NEIL3 play during DNA replication, model replication fork substrates were designed containing the oxidized bases at one of three specific sites relative to the fork. Results indicate that whilst specificity for 5- hydroxyuracil and thymine glycol was observed, NEIL1 acts preferentially on double-stranded DNA, including the damage upstream to the replication fork, whereas NEIL3 preferentially excises oxidized bases from single stranded DNA and within open fork structures. Thus, NEIL1 and NEIL3 act in concert to remove oxidized bases from the replication fork.

Interstrand cross-links (ICLs) are highly cytotoxic DNA lesions that block DNA replication and transcription by preventing strand separation. Previously, we demonstrated that the bacterial and human DNA glycosylases Nei and NEIL1 excise unhooked psoralen-derived ICLs in three-stranded DNA via hydrolysis of the glycosidic bond between the crosslinked base and deoxyribose sugar. Furthermore, NEIL3 from Xenopus laevis has been shown to cleave psoralen- and abasic site-induced ICLs in Xenopus egg extracts. Here we report that human NEIL3 cleaves psoralen-induced DNA-DNA cross-links in three-stranded and four-stranded DNA substrates to generate unhooked DNA fragments containing either an abasic site or a psoralen-thymine monoadduct. Furthermore, while Nei and NEIL1 also cleave a psoralen-induced four-stranded DNA substrate to generate two unhooked DNA duplexes with a nick, NEIL3 targets both DNA strands in the ICL without generating single-strand breaks. The DNA substrate specificities of these Nei-like enzymes imply the occurrence of long uninterrupted three- and four-stranded crosslinked DNA-DNA structures that may originate in vivo from DNA replication fork bypass of an ICL. In conclusion, the Nei-like DNA glycosylases unhook psoralen-derived ICLs in various DNA structures via a genuine repair mechanism in which complex DNA lesions can be removed without generation of highly toxic double-strand breaks.

The DNA mismatch repair (MMR) and base excision repair (BER) systems are important determinants of cellular toxicity following exposure to agents that cause oxidative DNA damage. To examine the interactions between these different repair systems, we examined whether toxicity, induced byt-BOOH and KBrO₃, differs in BER proficient (Mpg^+/+,Nth1^+/+) and deficient (Mpg^−/−,Nth1^−/−) mouse embryonic fibroblasts (MEFs) followingMsh2knockdown of between 79 and 88% using an shRNA expression vector.Msh2knockdown inNth1^+/+cells had no effect ont-BOOH and KBrO₃induced toxicity as assessed by an MTT assay; knockdown inNth1^−/−cells resulted in increased resistance tot-BOOH and KBrO₃, a result consistent with Nth1 removing oxidised pyrimidines.Msh2knockdown inMpg^+/+cells had no effect ont-BOOH toxicity but increased resistance to KBrO₃; inMpg^−/−cells,Msh2knockdown increased cellular sensitivity to KBrO₃but increased resistance to t-BOOH, suggesting a role forMpgin removing DNA damage induced by these agents. MSH2 dependent and independent pathways then determine cellular toxicity induced by oxidising agents. A complex interaction between MMR and BER repair systems, that is, exposure dependent, also exists to determine cellular toxicity.

OGG‐1 DNA glycosylase (OGG‐1) is an enzyme involved in DNA repair. It excises 7,8‐dihydro‐8‐oxoguanine, which is formed by oxidative damage of guanine. We have investigated the role of OGG‐1 in inflammation using three models of inflammation: endotoxic shock, diabetes, and contact hypersensitivity. We found that OGG‐1 ^−/− mice are resistant to endotoxin (lipopolysaccharide, LPS)‐induced organ dysfunction, neutrophil infiltration and oxidative stress, when compared with the response seen in wild‐type controls (OGG ^+/+ ). Furthermore, the deletion of the OGG‐1 gene was associated with decreased serum cytokine and chemokine levels and prolonged survival after LPS treatment. Type I diabetes was induced by multiple low‐dose streptozotocin treatment. OGG‐1 ^−/− mice were found to have significantly lower blood glucose levels and incidence of diabetes as compared with OGG‐1 ^+/+ mice. Biochemical analysis of the pancreas showed that OGG‐1 ^−/− mice had greater insulin content, indicative of a greater β‐cell mass coupled with lower levels of the chemokine MIP‐1α and Th1 cytokines IL‐12 and TNF‐α. Levels of protective Th2 cytokines, IL‐4 and IL‐10 were significantly higher in the pancreata of OGG‐1 ^−/− mice as compared with the levels measured in wild‐type mice. In the contact hypersensitivity induced by oxazolone, the OGG‐1 ^−/− mice showed reduced neutrophil accumulation, chemokine, and Th1 and Th2 cytokine levels in the ear tissue. The current studies unveil a role for OGG‐1 in the regulation of inflammation.

Ethenobases are promutagenic DNA adducts formed by some environmental carcinogens and products of endogenous lipid peroxidation. Mutation spectra in tumors induced in mice by urethane or its metabolite vinyl carbamate (Vcar) are compatible with 1,N(6)-ethenoadenine (epsilonA) being an initiating lesion in the development of these tumors. As alkylpurine-DNA-N-glycosylase (APNG) releases epsilonA from DNA in vitro, wild-type and APNG-/- C57Bl/6J mice were treated with Vcar and levels of epsilonA and 3,N(4)-ethenocytosine (epsilonC), which is not a substrate of APNG, were analyzed in liver and lung DNA. At 6 h after the last dose, levels of epsilonA were 1.6-fold higher in DNA from APNG-/- mice and subsequently persisted at higher levels for longer than in DNA from wild-type animals, confirming that epsilonA is released by APNG in vivo. In contrast, approximately 14-fold lower levels of epsilonC were induced by Vcar, and the kinetics of formation and persistence of epsilonC was similar in the two mouse strains. The carcinogenicity of Vcar was compared in APNG-/- and wild-type suckling mice given a single dose of Vcar (30 or 150 nmol/g). After 1 year, only mice in the high-dose group developed hepatocellular carcinoma; however, the incidence was not higher in APNG-/- mice. Although higher levels and increased persistence of epsilonA was observed in hepatic DNA from APNG-/- mice at 150 nmol/g Vcar, apoptosis and cell proliferation levels were similar in both strains of mice. This may explain why differences in epsilonA formation/persistence observed here did not result in higher susceptibility of APNG-/- mice to hepatocarcinogenesis.