Stable Interstrand Cross-Links Generated from the Repair of 1,N6-Ethenoadenine in DNA by α-Ketoglutarate/Fe(II)-Dependent Dioxygenase ALKBH2.

DNA cross-links severely challenge replication and transcription in cells, promoting senescence and cell death. In this paper, we report a novel type of DNA interstrand cross-link (ICL) produced as a side product during the attempted repair of 1,N6-ethenoadenine (εA) by human α-ketoglutarate/Fe(II)-dependent enzyme ALKBH2. This stable/nonreversible ICL was characterized by denaturing polyacrylamide gel electrophoresis analysis and quantified by high-resolution LC-MS in well-matched and mismatched DNA duplexes, yielding 5.7% as the highest level for cross-link formation. The binary lesion is proposed to be generated through covalent bond formation between the epoxide intermediate of εA repair and the exocyclic N6-amino group of adenine or the N4-amino group of cytosine residues in the complementary strand under physiological conditions. The cross-links occur in diverse sequence contexts, and molecular dynamics simulations rationalize the context specificity of cross-link formation. In addition, the cross-link generated from attempted εA repair was detected in cells by highly sensitive LC-MS techniques, giving biological relevance to the cross-link adducts. Overall, a combination of biochemical, computational, and mass spectrometric methods was used to discover and characterize this new type of stable cross-link both in vitro and in human cells, thereby uniquely demonstrating the existence of a potentially harmful ICL during DNA repair by human ALKBH2.

Effects of a Second Local DNA Damage Event on the Toxicity of the Human Carcinogen 4-Aminobiphenyl: A Molecular Dynamics Study of a Damaged DNA Structure.

Exposure of humans to carcinogenic aromatic amines (AAs) occurs daily. AAs are bioactivated in cells into products that attack DNA, primarily leading to N-linked C8-dG adducts. Previous work on DNA containing a single AA-derived adduct (monoadducted DNA) has shown a structure-function relationship between the damaged DNA conformation and cellular outcomes. However, relatively little is known about the conformation and biological outcomes of DNA containing two bulky adducts (diadducted DNA) in close proximity. To fill this current void in the literature, the present work uses quintuplet 0.5 µs MD simulations to understand the structural impact of DNA exposure to the potent bladder carcinogen 4-aminobiphenyl (ABP), which is found in cigarette smoke and select dyes, and results in the widely studied N-linked ABPdG adduct. Specifically, 18 unique DNA duplexes were investigated that contain one or two ABPdG adducts in the anti and/or syn glycosidic orientation(s) in all combinations of three G positions in the NarI mutation hotspot for AAs (5'-G1G2CG3CC). Monoadducted DNA displays sequence-dependent conformational heterogeneity, with the G1 site having the greatest anti preference, and highlights the range of helical structures associated with the syn lesion orientation [i.e., stacked (S), intercalated (I), and wedge (W) conformations]. Diadducted DNA results in interesting lesion separation effects on the conformational heterogeneity, including a greater anti preference for neighboring adducts (G1G2) and a greater syn preference for next-nearest neighbor damaged sites (G2G3) compared to monoadducted DNA. As a result, an increase in the number of ABPdG adducts changes the conformational heterogeneity of ABP-exposed DNA depending on the relative positions of the lesions and thereby could result in increased or decreased toxicity upon human exposure to elevated levels of ABP.

Impact of DNA Adduct Size, Number, and Relative Position on the Toxicity of Aromatic Amines: A Molecular Dynamics Case Study of ANdG- and APdG-Containing DNA Duplexes.

Human exposure to aromatic amines (AAs) can result in carcinogenic DNA adducts. To complement previous work geared toward understanding the mutagenicity of AA-derived adducts, which has almost exclusively studied (monoadducted) DNA containing a single lesion, the present work provides the first in-depth comparison of the structure of monoadducted and diadducted DNA duplexes. Specifically, molecular dynamics (MD) simulations were initially performed on DNA containing the nonmutagenic single-ringed N-(deoxyguanosin-8-yl)-aniline (ANdG) or the mutagenic four-ringed N-(deoxyguanosin-8-yl)-1-aminopyrene (APdG) lesion at G1, G2, or G3 in the AA deletion hotspot (5'-G1G2CG3CC) in the anti or syn glycosidic orientation (B/S duplex conformation). Subsequently, diadducted strands were assessed that span each combination of damaged sites (G1G2 (nearest neighbors), G2G3 (next-nearest neighbors), and G1G3 (two intervening nucleotides)) and anti/syn lesion glycosidic orientations. Despite other N-linked C8-dG adducts exhibiting sequence dependence conformational heterogeneity, a single ANdG or APdG lesion induces helical conformational homogeneity that is exclusively controlled by aryl moiety size. However, the preferred damaged DNA conformation can change upon the addition of a second adduct depending on lesion separation, with neighboring lesions stabilizing a nonmutagenic conformation and next-nearest damaged sites stabilizing a promutagenic conformation regardless of adduct size. As a result, diadducted DNA is found to adopt conformations that are unfavored for the corresponding monoadducted system, pointing to differential replication and repair outcomes for diadducted DNA compared to those for monoadducted DNA. Thus, although the toxicity of monoadducted DNA is most significantly dictated by lesion size, the toxicity can increase or decrease upon a second damaging event depending on lesion size and relative position. Overall, our work adds the number of lesions and their spatial separation to the growing list of factors that determine the structure and biological outcomes of adducted DNA.

Posttranscriptional modifications at the 37th position in the anticodon stem-loop of tRNA: structural insights from MD simulations.

Transfer RNA (tRNA) is the most diversely modified RNA. Although the strictly conserved purine position 37 in the anticodon stem-loop undergoes modifications that are phylogenetically distributed, we do not yet fully understand the roles of these modifications. Therefore, molecular dynamics simulations are used to provide molecular-level details for how such modifications impact the structure and function of tRNA. A focus is placed on three hypermodified base families that include the parent i6A, t6A, and yW modifications, as well as derivatives. Our data reveal that the hypermodifications exhibit significant conformational flexibility in tRNA, which can be modulated by additional chemical functionalization. Although the overall structure of the tRNA anticodon stem remains intact regardless of the modification considered, the anticodon loop must rearrange to accommodate the bulky, dynamic hypermodifications, which includes changes in the nucleotide glycosidic and backbone conformations, and enhanced or completely new nucleobase-nucleobase interactions compared to unmodified tRNA or tRNA containing smaller (m1G) modifications at the 37th position. Importantly, the extent of the changes in the anticodon loop is influenced by the addition of small functional groups to parent modifications, implying each substituent can further fine-tune tRNA structure. Although the dominant conformation of the ASL is achieved in different ways for each modification, the molecular features of all modified tRNA drive the ASL domain to adopt the functional open-loop conformation. Importantly, the impact of the hypermodifications is preserved in different sequence contexts. These findings highlight the likely role of regulating mRNA structure and translation.