Thomas E. Spratt

Interaction of Sirt3 with OGG1 contributes to repair of mitochondrial DNA and protects from apoptotic cell death under oxidative stress

Sirtuin 3 (Sirt3), a major mitochondrial NAD+-dependent deacetylase, targets various mitochondrial proteins for lysine deacetylation and regulates important cellular functions such as energy metabolism, aging, and stress response. In this study, we identified the human 8-oxoguanine-DNA glycosylase 1 (OGG1), a DNA repair enzyme that excises 7,8-dihydro-8-oxoguanine (8-oxoG) from damaged genome, as a new target protein for Sirt3. We found that Sirt3 physically associated with OGG1 and deacetylated this DNA glycosylase and that deacetylation by Sirt3 prevented the degradation of the OGG1 protein and controlled its incision activity. We further showed that regulation of the acetylation and turnover of OGG1 by Sirt3 played a critical role in repairing mitochondrial DNA (mtDNA) damage, protecting mitochondrial integrity, and preventing apoptotic cell death under oxidative stress. We observed that following ionizing radiation, human tumor cells with silencing of Sirt3 expression exhibited deteriorated oxidative damage of mtDNA, as measured by the accumulation of 8-oxoG and 4977 common deletion, and showed more severe mitochondrial dysfunction and underwent greater apoptosis in comparison with the cells without silencing of Sirt3 expression. The results reported here not only reveal a new function and mechanism for Sirt3 in defending the mitochondrial genome against oxidative damage and protecting from the genotoxic stress-induced apoptotic cell death but also provide evidence supporting a new mtDNA repair pathway.

An unprecedented nucleic acid capture mechanism for excision of DNA damage

DNA glycosylases that remove alkylated and deaminated purine nucleobases are essential DNA repair enzymes that protect the genome, and at the same time confound cancer alkylation therapy, by excising cytotoxic N3-methyladenine bases formed by DNA-targeting anticancer compounds. The basis for glycosylase specificity towards N3- and N7-alkylpurines is believed to result from intrinsic instability of the modified bases and not from direct enzyme functional group chemistry. Here we present crystal structures of the recently discovered Bacillus cereus AlkD glycosylase in complex with DNAs containing alkylated, mismatched and abasic nucleotides. Unlike other glycosylases, AlkD captures the extrahelical lesion in a solvent-exposed orientation, providing an illustration for how hydrolysis of N3- and N7-alkylated bases may be facilitated by increased lifetime out of the DNA helix. The structures and supporting biochemical analysis of base flipping and catalysis reveal how the HEAT repeats of AlkD distort the DNA backbone to detect non-Watson–Crick base pairs without duplex intercalation.

Glucuronidation of Tobacco-Specific Nitrosamines by UGT2B10

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is an important tobacco-specific nitrosamine (TSNA) in the etiology of tobacco-related cancers, and N-glucuronidation is an important mechanism of NNAL detoxification. In the present study, an analysis of the UDP-glucuronosyltransferases (UGTs) responsible for the N-glucuronidation of the TSNAs N′-nitrosonornicotine, N′-nitrosoanabasine, and N′-nitrosoanatabine was performed. Using human embryonic kidney 293 cells overexpressing UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, and UGT2B17, only UGT1A4 and UGT2B10 exhibited N-glucuronidating activity against these TSNAs. The KMs for UGT2B10 were 15 to 22-fold lower than those of UGT1A4 against the three TSNAs and were similar to those observed for microsomes prepared from human liver specimens. The overall activity of UGT2B10 was 3.6 to 27-fold higher than UGT1A4 against the three TSNAs as determined by Vmax/KM after normalization by levels of UGT2B10 versus UGT1A4 mRNA. Similarly high levels of activity were also observed for UGT2B10 against a fourth TSNA, NNAL, exhibiting a 6.3-fold lower KM and 3-fold higher normalized Vmax/KM than that observed for UGT1A4. Real-time polymerase chain reaction analysis showed that UGT2B10 was expressed at a level that, on average, was 26% higher than that observed for UGT1A4 in a screening of normal liver tissue specimens from 20 individual subjects. These data suggest that UGT2B10 is likely the most active UGT isoform in human liver for the N-glucuronidation of TSNAs.

In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

ABSTRACT Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a pregenomic RNA (pgRNA) by using a multifunctional polymerase (HP). A critical function of HP is its specific recognition of a viral RNA signal termed ε (Hε) located on pgRNA, which is required for specific packaging of pgRNA into viral nucleocapsids and initiation of viral reverse transcription. HP initiates reverse transcription by using itself as a protein primer (protein priming) and Hε as the obligatory template. We have purified HP from human cells that retained Hε binding activity in vitro. Furthermore, HP purified as a complex with Hε, but not HP alone, displayed in vitro protein priming activity. While the HP-Hε interaction in vitro and in vivo required the Hε internal bulge, but not its apical loop, and was not significantly affected by the cap-Hε distance, protein priming required both the Hε apical loop and internal bulge, as well as a short distance between the cap and Hε, mirroring the requirements for RNA packaging. These studies have thus established new HBV protein priming and RNA binding assays that should greatly facilitate the dissection of the requirements and molecular mechanisms of HP-Hε interactions, RNA packaging, and protein priming.

Yeast DNA polymerase η makes functional contacts with the DNA minor groove only at the incoming nucleoside triphosphate

DNA polymerase η (Polη) functions in the proficient bypass of a variety of DNA lesions. Relative to the replicative polymerases, Polη has a greater tolerance for distorted DNA geometries and possesses a low fidelity. X-ray crystal structures and studies with nucleotide analogs have implicated interactions with the DNA minor groove as being crucial for the high fidelity of replicative DNA polymerases. To determine whether Polη also makes such functionally important contacts with the DNA minor groove, here we examine the effects on Polη-catalyzed nucleotide incorporation when 3-deazaguanine, a base analog that lacks the ability to form minor-groove hydrogen bonds with the protein, is substituted for guanine at various positions in the DNA. From these studies, we conclude that Polη makes only a single functional contact with the DNA minor groove at the position of the incoming nucleotide; in this regard, Polη differs from high-fidelity DNA polymerases that are unable to replicate through DNA lesions. These results help explain the proficient ability of Polη for bypassing distorting DNA lesions.

Functional characterization of low-prevalence missense polymorphisms in the UDP-glucuronosyltransferase 1A9 gene.

The UDP-glucuronosyltransferase (UGT) 1A9 has been shown to play an important role in the detoxification of several carcinogens and clearance of anticancer and pain medications. The goal of the present study was to identify novel polymorphisms in UGT1A9 and characterize their effect on glucuronidation activity. The UGT1A9 gene was analyzed by direct sequencing of buccal cell genomic DNA from 90 healthy subjects. In addition to a previously identified single nucleotide polymorphism (SNP) at codon 33 resulting in an amino acid substitution (Met>Thr), two low-prevalence (<0.02) novel missense SNPs at codons 167 (Val>Ala) and 183 (Cys>Gly) were identified and are present in both white and African-American subjects. Glucuronidation activity assays using HEK293 cell lines overexpressing wild-type or variant UGT1A9 demonstrated that the UGT1A933Thr and UGT1A9183Gly variants exhibited differential glucuronidation activities compared with wild-type UGT1A9, but this was substrate-dependent. The UGT1A9167Ala variant exhibited levels of activity similar to those of wild-type UGT1A9 for all substrates tested. Whereas the wild-type and UGT1A933Thr and UGT1A9167Ala variants formed homodimers as determined by Western blot analysis of native polyacrylamide gels, the UGT1A9183Gly variant was incapable of homodimerization. These results suggest that several low-prevalence missense polymorphisms exist for UGT1A9 and that two of these (M33T and C183G) are functional. These results also suggest that although Cys183 is necessary for UGT1A9 homodimerization, the lack of capacity for UGT1A9 homodimerization is not sufficient to eliminate UGT1A9 activity.

Inhibition of Nuclear Factor-κB DNA Binding by Organoselenocyanates through Covalent Modification of the p50 Subunit

Most known chemopreventive agents including certain selenium compounds suppress the activation of the nuclear factor kappaB (NF-kappaB), but the mechanisms remain largely elusive. Toward this end, we initially showed that the inhibition of NF-kappaB DNA binding by benzyl selenocyanate (BSC) and 1,4-phenylenebis(methylene)selenocyanate (p-XSC) was reversed by the addition of DTT; this suggests the formation of DTT-reducible selenium-sulfur bonds between selenocyanate moieties and cysteine residues in NF-kappaB (p50) protein. Furthermore, the inhibitory effect of selenocyanates on NF-kappaB was not altered in the presence of physiologic level of reduced glutathione (1 mmol/L), suggesting that selenocyanates can also inhibit NF-kappaB in vivo. Using both matrix-assisted laser desorption/ionization-time of flight and tandem mass spectrometry fragmentation, we showed for the first time that the Cys(62) residue in the active site of NF-kappaB (p50) protein was modified by BSC through the formation of a selenium-sulfur bond. In addition, p-XSC-bound NF-kappaB (p50) protein was also detected by a radiotracer method. To provide further support, molecular models of both BSC and p-XSC positioned in the DNA binding pocket of the p50 were constructed through the covalent modification of Cys(62); the models reveal that DNA substrate could be hindered to enter its DNA binding region. This study shows for the first time that BSC and p-XSC may exert their chemopreventive activity, at least in part, by inhibiting NF-kappaB through covalent modification of Cys(62) of the p50 subunit of NF-kappaB.

Development of novel naphthalimide derivatives and their evaluation as potential melanoma therapeutics

Synthesis and anti-melanoma activity of various naphthalimide analogs, rationally modified by introducing isothiocyanate (ITC) and thiourea (TU) functionalities, found in well-known anti-cancer agents, is described. The structure-activity relationship comparison of the novel agents in inhibiting cancer cell growth was evaluated in various melanoma cell lines. Both ITC and TU analogs effectively inhibited cell viability and induced apoptosis in various human melanoma cells. Nitro substitution and increase in alkyl chain length, in general, enhanced the apoptotic activity of ITC derivatives. All the new compounds were well tolerated when injected intraperitoneal (i.p.) in mice at effective doses at which both the ITC and TU derivatives inhibited melanoma tumor growth in mice following i.p. xenograft. The nitro substituted naphthalimide-ITC derivative 3d was found to be the most effective in inducing apoptosis, and in inhibiting melanoma cell and tumor growth.

Association studies of excision repair cross-complementation group 1 (ERCC1) haplotypes with lung and head and neck cancer risk in a Caucasian population.

BACKGROUND The formation of bulky DNA adducts caused by diol epoxide derivatives of polycyclic aromatic hydrocarbons has been associated with tobacco-induced cancers, and inefficient repair of such adducts by the nucleotide excision repair (NER) system has been linked to increased risk of tobacco-induced lung and head and neck (H&N) cancers. The human excision repair cross-complementation group 1 (ERCC1) protein is essential for a functional NER system and genetic variation in ERCC1 may contribute to impaired DNA repair capacity and increased lung and H&N cancer risk. METHODS In order to comprehensively capture common genetic variation in the ERCC1 gene, Caucasian data from the International HapMap project was used to assess linkage disequilibrium and choose four tagSNPs (rs1319052, rs3212955, rs3212948, and rs735482) in the ERCC1 gene to genotype 452 lung cancer cases, 175 H&N cancer cases, and 790 healthy controls. Haplotypes were estimated using expectation maximization (EM) algorithm, and haplotype association with cancer was investigated using Haplo.stats software adjusting for known covariates. RESULTS The genotype and haplotype frequencies matched previous estimates from Caucasians. There was no significant difference in the prevalence of rs1319052, rs3212955, rs3212948, and rs735482 when comparing lung or H&N cancer cases with controls (p-values>0.05). Similarly, there was no association between ERCC1 haplotypes and lung or H&N cancer susceptibility in this Caucasian population (p-values>0.05). No associations were found when stratifying lung cancer cases by histology, sex, smoking status, or smoking intensity. CONCLUSIONS This study suggests that ERCC1 polymorphisms and haplotypes do not play a role in lung and H&N cancer susceptibility in Caucasians.

Discrimination between Right and Wrong Purine dNTPs by DNA Polymerase I from Bacillus stearothermophilus

We used a series of dATP and dGTP analogues to determine how DNA polymerase I from Bacillus stearothermophilus (BF), a prototypical A family polymerase, uses N-1, N(2), N-3, and N(6) of purine dNTPs to differentiate between right and wrong nucleotide incorporation. Altering any of these nitrogens had two effects. First, it decreased the efficiency of correct incorporation of the resulting dNTP analogue, with the loss of N-1 and N-3 having the most severe effects. Second, it dramatically increased the rate of misincorporation of the resulting dNTP analogues, with alterations in either N-1 or N(6) having the most severe impacts. Adding N(2) to dNTPs containing the bases adenine and purine increased the degree of polymerization opposite T but also tremendously increased the degree of misincorporation opposite A, C, and G. Thus, BF uses N-1, N(2), N-3, and N(6) of purine dNTPs both as negative selectors to prevent misincorporation and as positive selectors to enhance correct incorporation. Comparing how BF discriminates between right and wrong dNTPs with both B family polymerases and low-fidelity polymerases indicates that BF has chosen a unique solution vis-a-vis these other enzymes and, therefore, that nature has evolved at least three mechanistically distinct solutions.