Structural and Dynamic Features of the Recognition of 8-oxoguanosine Paired with an 8-oxoG-clamp by Human 8-oxoguanine-DNA Glycosylase.

8-oxoguanine (oxoG) is formed in DNA by the action of reactive oxygen species. As a highly mutagenic and the most common oxidative DNA lesion, it is an important marker of oxidative stress. Human 8-oxoguanine-DNA glycosylase (OGG1) is responsible for its prompt removal in human cells. OGG1 is a bifunctional DNA glycosylase with N-glycosylase and AP lyase activities. Aspects of the detailed mechanism underlying the recognition of 8-oxoguanine among numerous intact bases and its subsequent interaction with the enzyme's active site amino acid residues are still debated. The main objective of our work was to determine the effect (structural and thermodynamic) of introducing an oxoG-clamp in model DNA substrates on the process of 8-oxoG excision by OGG1. Towards that end, we used DNA duplexes modeling OGG1-specific lesions: 8-oxoguanine or an apurinic/apyrimidinic site with either cytidine or the oxoG-clamp in the complementary strand opposite to the lesion. It was revealed that there was neither hydrolysis of the N-glycosidic bond at oxoG nor cleavage of the sugar-phosphate backbone during the reaction between OGG1 and oxoG-clamp-containing duplexes. Possible structural reasons for the absence of OGG1 enzymatic activity were studied via the stopped-flow kinetic approach and molecular dynamics simulations. The base opposite the damage was found to have a critical effect on the formation of the enzyme-substrate complex and the initiation of DNA cleavage. The oxoG-clamp residue prevented the eversion of the oxoG base into the OGG1 active site pocket and impeded the correct convergence of the apurinic/apyrimidinic site of DNA and the attacking nucleophilic group of the enzyme. An obtained three-dimensional model of the OGG1 complex with DNA containing the oxoG-clamp, together with kinetic data, allowed us to clarify the role of the contact of amino acid residues with DNA in the formation of (and rearrangements in) the enzyme-substrate complex.

A Convenient Oligonucleotide Conjugation via Tandem Staudinger Reaction and Amide Bond Formation at the Internucleotidic Phosphate Position.

Staudinger reaction on the solid phase between an electronodeficit organic azide, such as sulfonyl azide, and the phosphite triester formed upon phosphoramidite coupling is a convenient method for the chemical modification of oligonucleotides at the internucleotidic phosphate position. In this work, 4-carboxybenzenesulfonyl azide, either with a free carboxy group or in the form of an activated ester such as pentafluorophenyl, 4-nitrophenyl, or pentafluorobenzyl, was used to introduce a carboxylic acid function to the terminal or internal internucleotidic phosphate of an oligonucleotide via the Staudinger reaction. A subsequent treatment with excess primary alkyl amine followed by the usual work-up, after prior activation with a suitable peptide coupling agent such as a uronium salt/1-hydroxybenzotriazole in the case of a free carboxyl, afforded amide-linked oligonucleotide conjugates in good yields including multiple conjugations of up to the exhaustive modification at each phosphate position for a weakly activated pentafluorobenzyl ester, whereas more strongly activated and, thus, more reactive aryl esters provided only single conjugations at the 5'-end. The conjugates synthesized include those with di- and polyamines that introduce a positively charged side chain to potentially assist the intracellular delivery of the oligonucleotide.

The Role of Key Amino Acids of the Human Fe(II)/2OG-Dependent Dioxygenase ALKBH3 in Structural Dynamics and Repair Activity toward Methylated DNA.

Non-heme dioxygenases of the AlkB family hold a unique position among enzymes that repair alkyl lesions in nucleic acids. These enzymes activate the Fe(II) ion and molecular oxygen through the coupled decarboxylation of the 2-oxoglutarate co-substrate to subsequently oxidize the substrate. ALKBH3 is a human homolog of E. coli AlkB, which displays a specific activity toward N1-methyladenine and N3-methylcytosine bases in single-stranded DNA. Due to the lack of a DNA-bound structure of ALKBH3, the basis of its substrate specificity and structure-function relationships requires further exploration. Here we have combined biochemical and biophysical approaches with site-directed mutational analysis to elucidate the role of key amino acids in maintaining the secondary structure and catalytic activity of ALKBH3. Using stopped-flow fluorescence spectroscopy we have shown that conformational dynamics play a crucial role in the catalytic repair process catalyzed by ALKBH3. A transient kinetic mechanism, which comprises the steps of the specific substrate binding, eversion, and anchoring within the DNA-binding cleft, has been described quantitatively by rate and equilibrium constants. Through CD spectroscopy, we demonstrated that replacing side chains of Tyr143, Leu177, and His191 with alanine results in significant alterations in the secondary structure content of ALKBH3 and decreases the stability of mutant proteins. The bulky side chain of Tyr143 is critical for binding the methylated base and stabilizing its flipped-out conformation, while its hydroxyl group is likely involved in facilitating the product release. The removal of the Leu177 and His191 side chains substantially affects the secondary structure content and conformational flexibility, leading to the complete inactivation of the protein. The mutants lacking enzymatic activity exhibit a marked decrease in antiparallel ß-strands, offset by an increase in the helical component.

Structure- and Content-Dependent Efficiency of Cas9-Assisted DNA Cleavage in Genome-Editing Systems.

Genome-editing systems, being some of the key tools of molecular biologists, represent a reasonable hope for progress in the field of personalized medicine. A major problem with such systems is their nonideal accuracy and insufficient selectivity. The selectivity of CRISPR-Cas9 systems can be improved in several ways. One efficient way is the proper selection of the consensus sequence of the DNA to be cleaved. In the present work, we attempted to evaluate the effect of formed non-Watson-Crick pairs in a DNA duplex on the efficiency of DNA cleavage in terms of the influence of the structure of the formed partially complementary pairs. We also studied the effect of the location of such pairs in DNA relative to the PAM (protospacer-adjacent motif) on the cleavage efficiency. We believe that the stabilization of the Cas9-sgRNA complex with a DNA substrate containing noncomplementary pairs is due to loop reorganization in the RuvC domain of the enzyme. In addition, PAM-proximal mismatches in the DNA substrate lower enzyme efficiency because the "seed" region is involved in binding and cleavage, whereas PAM-distal mismatches have no significant impact on target DNA cleavage. Our data suggest that in the case of short duplexes with mismatches, the stages of recognition and binding of dsDNA substrates by the enzyme determine the reaction rate and time rather than the thermodynamic parameters affected by the "unwinding" of DNA. The results will provide a theoretical basis for predicting the efficiency and accuracy of CRISPR-Cas9 systems at cleaving target DNA.

Thermodynamic Swings: How Ideal Complex of Cas9-RNA/DNA Forms.

Most processes of the recognition and formation of specific complexes in living systems begin with collisions in solutions or quasi-solutions. Then, the thermodynamic regulation of complex formation and fine tuning of complexes come into play. Precise regulation is very important in all cellular processes, including genome editing using the CRISPR-Cas9 tool. The Cas9 endonuclease is an essential component of the CRISPR-Cas-based genome editing systems. The attainment of high-specificity and -efficiency Cas9 during targeted DNA cleavage is the main problem that limits the practical application of the CRISPR-Cas9 system. In this study, we analyzed the thermodynamics of interaction of a complex's components of Cas9-RNA/DNA through experimental and computer simulation methods. We found that there is a small energetic preference during Cas9-RNA/DNA formation from the Cas9-RNA and DNA/DNA duplex. The small difference in binding energy is relevant for biological interactions and could be part of the sequence-specific recognition of double-stranded DNA by the CRISPR-Cas9 system.

Structural features of DNA polymerases ß and λ in complex with benzo[a]pyrene-adducted DNA cause a difference in lesion tolerance.

DNA polymerases ß (Pol ß) and λ (Pol λ) belong to one structural family (X family) and possess the same enzymatic activities. Nonetheless, these enzymes have differences in their catalytic efficiency and specificity. We have previously reported that these enzymes can bypass bulky benzo[a]pyrene-DNA adducts via translesion synthesis during gap-filling reactions, although efficiency and specificity are dependent on the reaction conditions and adduct conformation. In the present study, we analyzed structural features of Pols ß and λ complexed with a gapped DNA duplex containing either cis- or trans-benzo[a]pyrene-diol epoxide-N2-dG (BP-dG) using molecular dynamics simulations. It was found that the most pronounced structural difference lies in the positioning of the trans-BP-dG residue relative to secondary structures of the protein; this dissimilarity may explain the differences between Pols ß and λ in gap-filling/translesion synthesis. In the case of Pol ß, trans-BP-dG turned out to be positioned parallel to the α-helix and ß-sheet. In the Pol λ complex, trans-BP-dG is perpendicular to the α-helix. This difference persisted throughout the molecular dynamics trajectory. Selectivity for the BP-dG isomers remained after a deletion of noncatalytic domains of Pol λ. Modeling of Pol λ or ß complexes with cis-BP-dG-containing DNA in the presence of Mn2+ either at both metal-binding sites or at the catalytic site only revealed that for both enzymes, the model of the complex containing both Mg2+ and Mn2+ is stabler than that containing two Mn2+ ions. This observation may reflect a shared property of these enzymes: the preference for Mn2+ in terms of catalysis and for Mg2+ regarding triphosphate coordination during the translesion reaction.

Probing the Dynamics of Streptococcus pyogenes Cas9 Endonuclease Bound to the sgRNA Complex Using Hydrogen-Deuterium Exchange Mass Spectrometry.

The Cas9 endonuclease is an essential component of the CRISPR-Cas-based genome editing tools. The attainment of high specificity and efficiency of Cas9 during targetted DNA cleavage is the main problem that limits the clinical application of the CRISPR-Cas9 system. A deep understanding of the Cas9 mechanism and its structural-functional relationships is required to develop strategies for precise gene editing. Here, we present the first attempt to describe the solution structure of Cas9 from S. pyogenes using hydrogen-deuterium exchange mass spectrometry (HDX-MS) coupled to molecular dynamics simulations. HDX data revealed multiple protein regions with deuterium uptake levels varying from low to high. By analysing the difference in relative deuterium uptake by apoCas9 and its complex with sgRNA, we identified peptides involved in the complex formation and possible changes in the protein conformation. The REC3 domain was shown to undergo the most prominent conformational change upon enzyme-RNA interactions. Detection of the HDX in two forms of the enzyme provided detailed information about changes in the Cas9 structure induced by sgRNA binding and quantified the extent of the changes. The study demonstrates the practical utility of HDX-MS for the elucidation of mechanistic aspects of Cas9 functioning.

Dataset for dynamics and conformational changes in human NEIL2 protein analyzed by integrative structural biology approach.

This work presents new data on human endonuclease VIII-like 2 protein (hNEIL2), a part of DNA glycosylases of the helix-two-turn-helix structural superfamily. While X-ray structure of oNEIL2 (opossum Monodelphis) was resolved partially [1], the structure of hNEIL2 has not yet been determined. This data article describes a powerful combination of hydrogen-deuterium exchange mass spectrometry, homology modeling, and molecular dynamics simulations for protein conformational dynamics analysis. The data supplied in this work are related to the research article entitled "Dynamics and Conformational Changes in Human NEIL2 DNA Glycosylase Analyzed by Hydrogen/Deuterium Exchange Mass Spectrometry".

Dynamics and Conformational Changes in Human NEIL2 DNA Glycosylase Analyzed by Hydrogen/Deuterium Exchange Mass Spectrometry.

Base excision DNA repair (BER) is necessary for removal of damaged nucleobases from the genome and their replacement with normal nucleobases. BER is initiated by DNA glycosylases, the enzymes that cleave the N-glycosidic bonds of damaged deoxynucleotides. Human endonuclease VIII-like protein 2 (hNEIL2), belonging to the helix-two-turn-helix structural superfamily of DNA glycosylases, is an enzyme uniquely specific for oxidized pyrimidines in non-canonical DNA substrates such as bubbles and loops. The structure of hNEIL2 has not been solved; its closest homologs with known structures are NEIL2 from opossum and from giant mimivirus. Here we analyze the conformational dynamics of free hNEIL2 using a combination of hydrogen/deuterium exchange mass spectrometry, homology modeling and molecular dynamics simulations. We show that a prominent feature of vertebrate NEIL2 - a large insert in its N-terminal domain absent from other DNA glycosylases - is unstructured in solution. It was suggested that helix-two-turn-helix DNA glycosylases undergo open-close transition upon DNA binding, with the large movement of their N- and C-terminal domains, but the open conformation has been elusive to capture. Our data point to the open conformation as favorable for free hNEIL2 in solution. Overall, our results are consistent with the view of hNEIL2 as a conformationally flexible protein, which may be due to its participation in the repair of non-canonical DNA structures and/or to the involvement in functional and regulatory protein-protein interactions.