Mark Lukin

DNA adducts of aristolochic acid II: total synthesis and site-specific mutagenesis studies in mammalian cells

Aristolochic acids I and II (AA-I, AA-II) are found in all Aristolochia species. Ingestion of these acids either in the form of herbal remedies or as contaminated wheat flour causes a dose-dependent chronic kidney failure characterized by renal tubulointerstitial fibrosis. In ∼50% of these cases, the condition is accompanied by an upper urinary tract malignancy. The disease is now termed aristolochic acid nephropathy (AAN). AA-I is largely responsible for the nephrotoxicity while both AA-I and AA-II are genotoxic. DNA adducts derived from AA-I and AA-II have been isolated from renal tissues of patients suffering from AAN. We describe the total synthesis, de novo, of the dA and dG adducts derived from AA-II, their incorporation site-specifically into DNA oligomers and the splicing of these modified oligomers into a plasmid construct followed by transfection into mouse embryonic fibroblasts. Analysis of the plasmid progeny revealed that both adducts blocked replication but were still partly processed by DNA polymerase(s). Although the majority of coding events involved insertion of correct nucleotides, substantial misincorporation of bases also was noted. The dA adduct is significantly more mutagenic than the dG adduct; both adducts give rise, almost exclusively, to misincorporation of dA, which leads to AL-II-dA→T and AL-II-dG→T transversions.

Structure and stability of DNA containing an aristolactam II-dA lesion: implications for the NER recognition of bulky adducts

Aristolochic acids I and II are prevalent plant toxicants found in the Aristolochiaceae plant family. Metabolic activation of the aristolochic acids leads to the formation of a cyclic N-hydroxylactam product that can react with the peripheral amino group of purine bases generating bulky DNA adducts. These lesions are mutagenic and established human carcinogens. Interestingly, although AL-dG adducts progressively disappear from the DNA of laboratory animals, AL-dA lesions has lasting persistence in the genome. We describe here NMR structural studies of an undecameric duplex damaged at its center by the presence of an ALII-dA adduct. Our data establish a locally perturbed double helical structure that accommodates the bulky adduct by displacing the counter residue into the major groove and stacking the ALII moiety between flanking bases. The presence of the ALII-dA perturbs the conformation of the 5′-side flanking base pair, but all other pairs of the duplex adopt standard conformations. Thermodynamic studies reveal that the lesion slightly decreases the energy of duplex formation in a sequence-dependent manner. We discuss our results in terms of its implications for the repair of ALII-dA adducts in mammalian cells.

Organization of the human mitochondrial transcription initiation complex

Initiation of transcription in human mitochondria involves two factors, TFAM and TFB2M, in addition to the mitochondrial RNA polymerase, POLRMT. We have investigated the organization of the human mitochondrial transcription initiation complex on the light-strand promoter (LSP) through solution X-ray scattering, electron microscopy (EM) and biochemical studies. Our EM results demonstrate a compact organization of the initiation complex, suggesting that protein–protein interactions might help mediate initiation. We demonstrate that, in the absence of DNA, only POLRMT and TFAM form a stable interaction, albeit one with low affinity. This is consistent with the expected transient nature of the interactions necessary for initiation and implies that the promoter DNA acts as a scaffold that enables formation of the full initiation complex. Docking of known crystal structures into our EM maps results in a model for transcriptional initiation that strongly correlates with new and existing biochemical observations. Our results reveal the organization of TFAM, POLRMT and TFB2M around the LSP and represent the first structural characterization of the entire mitochondrial transcriptional initiation complex.

The EM structure of human DNA polymerase γ reveals a localized contact between the catalytic and accessory subunits

We used electron microscopy to examine the structure of human DNA pol γ, the heterotrimeric mtDNA replicase implicated in certain mitochondrial diseases and aging models. Separate analysis of negatively stained preparations of the catalytic subunit, pol γA, and of the holoenzyme including a dimeric accessory factor, pol γB2, permitted unambiguous identification of the position of the accessory factor within the holoenzyme. The model explains protection of a partial chymotryptic cleavage site after residue L549 of pol γA upon binding of the accessory subunit. This interaction region is near residue 467 of pol γA, where a disease‐related mutation has been reported to impair binding of the B subunit. One pol γB subunit dominates contacts with the catalytic subunit, while the second B subunit is largely exposed to solvent. A model for pol γ is discussed that considers the effects of known mutations in the accessory subunit and the interaction of the enzyme with DNA.

Catalytic transformations of supercoiled DNA as studied by flow linear dichroism technique

A catalytic turnover of supercoiled DNA (scDNA) transformation mediated by topoisomerases leads to changes in the linking number (Lk) of the polymeric substrate by 1 or 2 per cycle. As a substrate of the topoisomerization reaction it is chemically identical to its product; even a single catalytic event results in the quantum leap in the scDNA topology. Non‐intrusive continuous assay to measure the kinetics of the scDNA topoisomerization was performed. The development of such a technique was hindered because of multiple DNA species of intermediate topology present in the reaction mixture. The interrelation of DNA topology, its hydrodynamics, and optical anisotropy enable us to use the flow linear dichroism technique (FLD) for continuous monitoring of the scDNA topoisomerization reaction. This approach permits us to study the kinetics of DNA transformation catalyzed by eukaryotic topoisomerases I and II, as well as mechanistic characteristics of these enzymes and their interactions with anticancer drugs. Moreover, FLD assay can be applied to any enzymatic reaction that involves scDNA as a substrate. It also provides a new way of screening drugs dynamically and is likely to be potent in various biomedical applications.

Incorporation of 3-aminobenzanthrone into 2'-deoxyoligonucleotides and its impact on duplex stability.

3-Nitrobenzanthrone (3NBA), an environmental pollutant and potent mutagen, causes DNA damage via the reaction of its metabolically activated form with the exocyclic amino groups of purines and the C-8 position of guanine. The present work describes a synthetic approach to the preparation of oligomeric 2′-deoxyribonucleotides containing a 2-(2′-deoxyguanosin-N2-yl)-3-aminobenzanthrone moiety, one of the major DNA adducts found in tissues of living organisms exposed to 3NBA. The NMR spectra indicate that the damaged oligodeoxyribonucleotide is capable of forming a regular double helical structure with the polyaromatic moiety assuming a single conformation at room temperature; the spectra suggest that the 3ABA moiety resides in the duplex minor groove pointing toward the 5′-end of the modified strand. Thermodynamic studies show that the dG(N2)-3ABA lesion has a stabilizing effect on the damaged duplex, a fact that correlates well with the long persistence of this damage in living organisms.

Stereoselective Nucleoside Deuteration for NMR Studies of DNA

A procedure has been elaborated for stereoselective deuterium substitution of one of the diastereotopic 5′-protons in 2′-deoxynucleotides. The synthetic scheme uses the reduction of the 5-oxosugar derivative with deuterated Alpine-Borane. The resulting deuterosugar is converted into pyrimidine nucleosides and incorporated into DNA using standard protocols. Comparison of two-dimensional NMR spectra of the fully protonated and partially deuterated duplexes allowed us to assign diastereotopic 5′ protons, increasing the number of experimental restraints used for structure determination.

NMR structure of duplex DNA containing the alpha-OH-PdG.dA base pair: a mutagenic intermediate of acrolein.

Acrolein, a cell metabolic product and main component of cigarette smoke, reacts with DNA generating alpha-OH-PdG lesions, which have the ability to pair with dATP during replication thereby causing G to T transversions. We describe the solution structure of an 11-mer DNA duplex containing the mutagenic alpha-OH-PdG.dA base pair intermediate, as determined by solution nuclear magnetic resonance (NMR) spectroscopy and retrained molecular dynamics (MD) simulations. The NMR data support a mostly regular right-handed helix that is only perturbed at its center by the presence of the lesion. Undamaged residues of the duplex are in anti orientation, forming standard Watson-Crick base pairs alignments. Duplication of proton signals at and near the damaged base pair reveals the presence of two enantiomeric duplexes, thus establishing the exocyclic nature of the lesion. The alpha-OH-PdG adduct assumes a syn conformation pairing to its partner dA base that is protonated at pH 6.6. The three-dimensional structure obtained by restrained molecular dynamics simulations show hydrogen bond interactions that stabilize alpha-OH-PdG in a syn conformation and across the lesion containing base pair. We discuss the implications of the structures for the mutagenic bypass of acrolein lesions.

Genotoxic and cytotoxic effects of the environmental pollutant 3-nitrobenzanthrone on bladder cancer cells

3-Nitrobenzanthrone (3-NBA), a potential human carcinogen, is present in diesel exhaust. The main metabolite of 3-NBA, 3-aminobenzanthrone, was detected in urine of miners occupationally exposed to diesel emissions. Environmental and occupational factors play an important role in development of bladder cancer (BC), one of the most frequent malignancies. It is expected that exposure of urothelium to 3-NBA and its metabolites may induce BC initiation and/or progression. To test this hypothesis, we studied geno- and cytotoxicity of 3-NBA using an in vitro BC model. 3-NBA induced higher levels of DNA adducts, reactive oxygen species and DNA breaks in aggressive T24 cells than in more differentiated RT4 cells. To understand the nature of this difference we examined the role of several enzymes that were identified as 3-NBA bio activators. However, the difference in DNA adduct formation cannot be directly linked to the different activity of any of the examined enzymes. Conversely, the difference of tested cell lines in p53 status can partly explain the distinct levels of 3-NBA-DNA adducts and DNA damage induced by 3-NBA. Therefore, we assume that more aggressive T24 cells are more predisposed for DNA adduct formation, DNA damage and, possibly, mutations and as a result further tumorigenesis.

138 2′-Deoxyguanine analogs which enhance hybridization oligonucleotides with complementary DNA

et al., 2014). Here we present the results of the SER titration via an excess of competing oligonucleotide, according to (Reynaldo, Vologodskij, Neri, & Lyamichev, 2000). The sensitivity of the HG122 promoted SER to limited heterology between the reaction substrates was also estimated. As seen from the Figure, both HG122 and Rec A protein promote SER. In both cases, SER exhibits decreased sensibility to homology violation unlike SER promoted by other agents, such as linker histone H1, or mammalian meiotic system protein Hop2. The latter exhibit the same sensitivity to heterology as that of spontaneous SER at increased temperature (Bocharova, Smirnova, & Volodin, 2012; Pezza et al., 2014). Thus, 1,3-diazaadamantane derivative HG122 acts similar to recombinase Rec A family. This ligand facilitates SER, destabilizes the DNA duplex and appreciably reduces SER sensitivity to heterology between SER substrates. Therefore, compounds of this class are worthy models for further study of different aspects of SER mechanisms.