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The majority of base pairs in double-stranded DNA exist in the canonical Watson Crick geometry. However, they can also adopt alternate Hoogsteen conformations in various complexes of DNA with proteins and small molecules, which are key for biological function and mechanism. While detection of Hoogsteen base pairs in large DNA complexes and assemblies poses considerable challenges for traditional structural biology techniques, we show here that multidimensional dynamic nuclear polarization–enhanced solid-state NMR can serve as a unique spectroscopic tool for observing and distinguishing Watson-Crick and Hoogsteen base pairs in a broad range of DNA systems based on characteristic NMR chemical shifts and internuclear dipolar couplings. We illustrate this approach using a model 12-mer DNA duplex, free and in complex with the antibiotic echinomycin, which features two central adenine-thymine base pairs with Watson-Crick and Hoogsteen geometry, respectively, and subsequently extend it to the ∼200 kDa Widom 601 DNA nucleosome core particle. [ABSTRACT FROM AUTHOR]

Despite the DNA duplex being central to biological functions, many intricacies of this molecule, including the dynamic nature of mismatched base pairing, are still unknown. The unique conformations adopted by DNA mismatches can provide insight into the forces at play between nucleotides. Moreover, DNA‐binding proteins apply their own individualized steric and electrochemical influences on the nucleotides that they interact with, further altering base‐pairing conformations. Here, seven X‐ray crystallographic structures of the human nuclease apurinic/apyrimidinic (AP) endonuclease 1 (APE1) in complex with its substrate target flanked by a 5′ mismatch are reported. The structures reveal how APE1 influences the conformations of a variety of different mismatched base pairs. Purine–purine mismatches containing a guanine are stabilized by a rotation of the guanine residue about the N‐glycosidic bond to utilize the Hoogsteen edge for hydrogen bonding. Interestingly, no rotation of adenine, the other purine, is observed. Mismatches involving both purine and pyrimidine bases adopt wobble conformations to accommodate the mismatch. Pyrimidine–pyrimidine mismatches also wobble; however, the smaller profile of a pyrimidine base results in a gap between the Watson–Crick faces that is reduced by a C1′–C1′ compression. These results advance our understanding of mismatched base pairing and the influence of a bound protein. [ABSTRACT FROM AUTHOR]

We have reported the interaction of two flavonoids namely quercetin (Q) and morin (M) with double stranded poly(A).poly(U) (herein after A.U) and triple stranded poly(U).poly(A)*poly(U) (herein after U.A*U, dot represents the Watson–Crick and asterisk represents Hoogsteen base pairing respectively) in this article. It has been observed that relative positions of hydroxyl groups on the B-ring of the flavonoids affect the stabilization of RNA. The double strand as well as the triple strand of RNA-polymers become more stabilized in presence of Q, however both the duplex and triplex remain unaffected in presence of M. The presence of catechol moiety on the B-ring of Q is supposed to be responsible for the stabilization. Moreover, after exploiting a series of biophysical experiments, it has been found that, triple helical RNA becomes more stabilized over its parent duplex in presence of Q. Fluorescence quenching, viscosity measurement and helix melting results establish the fact that Q binds with both forms of RNA through the mode of intercalation while M does not bind at all to either forms of RNA. [ABSTRACT FROM AUTHOR]

Normal cellular homeostasis depends on tight regulation of gene expression, which requires the modulation of transcription factors' DNA-binding specificity. That said, the mechanisms that allow transcription factors to distinguish between closely related response elements following different cellular signals are not fully understood. In the tumor suppressor protein p53, acetylation of loop L1 residue Lys120 within the DNA-binding domain has been shown to promote the transcription of proapoptotic genes such as bax . Here, we report the crystal structures of Lys120-acetylated p53 DNA-binding domain in complex with a consensus response element and with the natural BAX response element. Our structural analyses reveal that Lys120 acetylation expands the conformational space of loop L1 in the DNA-bound state. Loop L1 flexibility is known to increase p53's DNA-binding specificity, and Lys120-acetylation-dependent conformational changes in loop L1 enable the formation of sequence-dependent DNA-binding modes for p53. Furthermore, binding to the natural BAX response element is accompanied by global conformational changes, deformation of the DNA helical structure, and formation of an asymmetric tetrameric complex. Based on these findings, we suggest a model for p53's Lys120 acetylation-dependent DNA-binding mode. [ABSTRACT FROM AUTHOR]

A new intercalating nucleic acid monomerXwas obtained in high yield starting from alkylation of 4-iodophenol with (S)-(+)-2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethanol under Mitsunobu conditions followed by hydrolysis with 80% aqueous acetic acid to give a diol which was coupled under Sonogashira conditions with trimethylsilylacetylene (TMSA) to achieve the TMS protected (S)-4-(4-((trimethylsilyl)ethynyl)phenoxy)butane-1,2-diol. Tetrabutylammonium flouride was used to remove the silyl protecting group to obtain (S)-4-(4-ethynylphenoxy)butane-1,2-diol which was coupled under Sonogashira conditions with 2-(9-bromo-6H-indolo[2,3-b]quinoxalin-6-yl)-N,N-dimethylethanamine to achieve (S)-4-(4-((6-(2-(dimethylamino)ethyl)-6H-indolo[2,3-b]quinoxalin-9-yl)ethynyl)phenoxy)butane-1,2-diol. This compound was tritylated with 4,4′-dimethoxytrityl chloride followed by treatment with 2-cyanoethyltetraisopropylphosphordiamidite in the presence of N,N′-diisopropyl ammonium tetrazolide to afford the corresponding phosphoramidite. This phosphoramidite was used to insert the monomerXinto an oligonucleotide which was used for thermal denaturation studies of a corresponding parallel triplex. [ABSTRACT FROM PUBLISHER]

Abstract: The effects of UV synchrotron radiation on deoxyribonucleic acid (DNA) cast films have been systematically investigated by vacuum ultraviolet and infrared spectrophotometry as a function of irradiation time. Cast DNA films exposed at 140 nm (8.85 eV) for different irradiation times, revealed consistent changes in their VUV spectra which indicate a decrease of thymine groups and an increase of π → π* transition spectral signature associated with the Cf the open sugar chain. This result was corroborated by a decrease in Cing vibration at 1061 cm−1 observed in the infrared spectra. Both these results are consistent with the creation of single strand breaks in the deoxyribose component of DNA molecule and a decrease in the phosphate groups. It was also shown that UV radiation is effective in damaging the thymine groups involved in Hoogsteen base pairing with adenine. The analysis of the infrared data suggests that the usual spectroscopic fingerprints of DNA denaturation are not necessarily a reliable measure of DNA damage. [Copyright &y& Elsevier]

Summary: DNA polymerase (pol) ι, a member of the mammalian Y-family of DNA polymerases involved in translesion DNA synthesis, has been previously suggested to peculiarly utilize Hoogsteen base pairing for DNA synthesis opposite template purines, unlike pols η and κ, which utilize Watson–Crick (W–C) base pairing. To investigate the possible roles of Hoogsteen, W–C, and wobble base-pairing modes in the selection of nucleotides opposite template pyrimidines by human pol ι, we carried out kinetic analyses of incorporation of modified purine nucleoside triphosphates including 7-deazapurines, inosine, 2-aminopurine, 2,6-diaminopurine, and 6-chloropurine, which affect H-bonding in base-pair formation opposite template pyrimidines. Carbon substitution at the N7 atom of purine nucleoside triphosphates, which disrupts Hoogsteen base pairing, only slightly inhibited DNA synthesis opposite template pyrimidines by pol ι, which was not substantially different from human pols η and κ. Opposite template T, only the relative wobble stabilities (inferred from the potential numbers of H-bonding, steric, and electrostatic interactions but not measured) of base pairs were positively correlated to the relative efficiencies of nucleotide incorporation by pol ι but not the relative W–C or Hoogsteen stabilities, unlike pols η and κ. In contrast, opposite C, only the relative W–C stabilities of base pairs were positively correlated to the relative efficiencies of nucleotide incorporation by pol ι, as with pols η and κ. These results suggest that pol ι might not indispensably require Hoogsteen base pairing for DNA synthesis opposite pyrimidines but rather might prefer wobble base pairing in the selection of nucleotides opposite T and W–C base pairing opposite C. [Copyright &y& Elsevier]

UV-light-induced cyclobutane pyrimidine dimers (CPDs) present a severe block to synthesis by replicative DNA polymerases (Pols), whereas Polη promotes proficient and error-free replication through CPDs. Although the archael Dpo4, which, like Polη, belongs to the Y family of DNA Pols, can also replicate through a CPD, it is much less efficient than Polη. The x-ray crystal structure of Dpo4 complexed with either the 3'-thymine (T) or the 5' T of a cis-syn TT dimer has indicated that, whereas the 3' T of the dimer forms a Watson-Crick base pair with the incoming dideoxy ATP, the 5' T forms a Hoogsteen base pair with the dideoxy ATP in syn conformation. Based upon these observations, a similar mechanism involving Hoogsteen base pairing of the 5' T of the dimer with the incoming A has been proposed for Polη. Here we examine the mechanisms of CPD bypass by Dpo4 and Polη using nucleotide analogs that specifically disrupt the Hoogsteen or Watson-Crick base pairing. Our results show that both Dpo4 and Polη incorporate dATP opposite the 5' T of the CPD via Watson-Crick base pairing and not by Hoogsteen base pairing. Furthermore, opposite the 3' T of the dimer, the two Pols differ strikingly in the mechanisms of dATP incorporation, with Dpo4 incorporating opposite an abasic-like intermediate and Polη using the normal watson-Crick base pairing. These observations have important implications for the mechanisms used for the inefficient vs. efficient bypass of CPDs by DNA Pols. [ABSTRACT FROM AUTHOR]

Template-directed oligomerization of an activated derivative of 3-isoadenosine 5′-phosphate (piA) on polyuridylic acid [poly(U)] was studied. The reaction of ImpiA is more efficient than the corresponding reaction of ImpA, and produces 3′-5′-linked oligomers while the reaction of ImpA gives only 2′-5′-linked oligomers. The base pairing between piA and poly(U) in this system is probably of the Hoogsteen type (involving the 6-amino group and N7 of 3-isoadenosine) rather than of the Watson-Crick type. [ABSTRACT FROM AUTHOR]

In a previous study, we showed that replication through the N1-methyl-deoxyadenosine (1-MeA) adduct in human cells is mediated via three different Polι/Polθ, Polη, and Polζ-dependent pathways. Based on biochemical studies with these Pols, in the Polι/Polθ pathway, we inferred a role for Polι in the insertion of a nucleotide (nt) opposite 1-MeA and of Polθ in extension of synthesis from the inserted nt; in the Polη pathway, we inferred that this Pol alone would replicate through 1-MeA; in the Polζ pathway, however, the Pol required for inserting an nt opposite 1-MeA had remained unidentified. In this study, we provide biochemical and genetic evidence for a role for Polλ in inserting the correct nt T opposite 1-MeA, from which Polζ would extend synthesis. The high proficiency of purified Polλ for inserting a T opposite 1-MeA implicates a role for Polλ-which normally uses W-C base pairing for DNA synthesis-in accommodating 1-MeA in a syn confirmation and forming a Hoogsteen base pair with T. The potential of Polλ to replicate through DNA lesions by Hoogsteen base pairing adds another novel aspect to Polλ's role in translesion synthesis in addition to its role as a scaffolding component of Polζ. We discuss how the action mechanisms of Polλ and Polζ could be restrained to inserting a T opposite 1-MeA and extending synthesis thereafter, respectively., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The action mechanisms revealed by the biochemical and structural analyses of replicative and translesion synthesis (TLS) DNA polymerases (Pols) are retained in their cellular roles. In this regard, DNA polymerase θ differs from other Pols in that whereas purified Polθ misincorporates an A opposite 1, N 6 conformation for Hoogsteen base pairing with the correct nucleotide. The evidence that a DNA polymerase can configure its active site entirely differently in human cells than in the purified Pol establishes a new paradigm for DNA polymerase function.syn conformation for Hoogsteen base pairing with the correct nucleotide. The evidence that a DNA polymerase can configure its active site entirely differently in human cells than in the purified Pol establishes a new paradigm for DNA polymerase function., (© 2020 Yoon et al.)

Here we show that translesion synthesis (TLS) opposite 1,N 6 -ethenodeoxyadenosine (εdA), which disrupts Watson-Crick base pairing, occurs via Polι/Polζ-, Rev1-, and Polθ-dependent pathways. The requirement of Polι/Polζ is consistent with the ability of Polι to incorporate nucleotide opposite εdA by Hoogsteen base pairing and of Polζ to extend synthesis. Rev1 polymerase and Polθ conduct TLS opposite εdA via alternative error-prone pathways. Strikingly, in contrast to extremely error-prone TLS opposite εdA by purified Polθ, it performs predominantly error-free TLS in human cells. Reconfiguration of the active site opposite εdA would provide Polθ the proficiency for error-free TLS in human cells., (© 2019 Yoon et al.; Published by Cold Spring Harbor Laboratory Press.)