Unraveling the Base Excision Repair Mechanism of Human DNA Glycosylase

Human DNA glycosylase, hOGG1, is known to perform DNA repair by cleaving oxidized guanine (8OG) from the DNA. Despite numerous experimental and theoretical investigations, the underlying selective molecular mechanism has remained a mystery. Here we present a mechanism that explains how hOGG1’s catalytic pocket is able to host an undamaged guanine base, but is not able to cleave it from the DNA. Using linear-scaling quantum mechanics/molecular mechanics (QM/MM) techniques with more than 500 atoms in the QM-region, we have investigated previously proposed mechanisms that all rely on protonating the 8OG nucleobase. We have found that the repair mechanisms propagated in the literature to this date are not capable of differentiating between the G and 8OG nucleobase. Besides this nonselectivity, they also involve reaction barriers that are too high, hence rendering the corresponding reaction intermediates inaccessible. Instead, we present a completely different reaction mechanism, where hOGG1 initially targets the ribose moiety of the substrate and cleaves the glycosidic bond at the very last stage. Our ribose-protonated repair mechanism is not only energetically more preferable, but also explains the selectivity utilized by hOGG1 to block processing a guanine base

On the Photophysics of Artificial Blue-Light Photoreceptors: An Ab Initio Study on a Flavin-Based Dye Dyad at the Level of Coupled-Cluster Response Theory

The photophysical behavior of a phenothiazine−phenyl−isoalloxazine dye dyad, a model system for blue-light photoreceptors functioning on the basis of photoinduced electron transfer, was investigated by employing a combination of time-dependent density functional and coupled-cluster response theory. A conical intersection between a “bright” locally excited and a “dark” charge-transfer state was found in the low-energy region of the corresponding potential energy surfaces. We propose that, for the solvated dyad, this conical intersection is responsible for the experimentally observed fast fluorescence quenching in that system

A Conclusive Mechanism of the Photoinduced Reaction Cascade in Blue Light Using Flavin Photoreceptors

On the basis of extensive first-principle calculations within the framework of quantum mechanics/molecular mechanics (QM/MM), a conclusive mechanism for the formation of the signaling state of blue light using flavin (BLUF) domain proteins is proposed which is compatible with the experimental data presently available. Time-dependent density functional, as well as advanced coupled cluster response theory was employed for the QM part in order to describe the relevant excited states. One of the key residues involved in the mechanism is the glutamine adjacent to the flavin chromophore. The reaction cascade, triggered by the initial photoexcitation of the flavin chromophore, involves isomerization of this residue but no rotation as assumed previously. The fact that only the environment, but not the flavin chromophore by itself, is chemically transformed along the individual steps of the mechanism is unique for biological photoreceptors. The final isomer of the glutamine tautomer, i.e., the imidic acid, is further stabilized by the interchange of a methionine residue in the binding pocket with a tryptophan residue. The flip of these two residues might be the trigger for the large conformational change of this protein which is consequently transmitted as the signal to the biological environment

A Conclusive Mechanism of the Photoinduced Reaction Cascade in Blue Light Using Flavin Photoreceptors

On the basis of extensive first-principle calculations within the framework of quantum mechanics/molecular mechanics (QM/MM), a conclusive mechanism for the formation of the signaling state of blue light using flavin (BLUF) domain proteins is proposed which is compatible with the experimental data presently available. Time-dependent density functional, as well as advanced coupled cluster response theory was employed for the QM part in order to describe the relevant excited states. One of the key residues involved in the mechanism is the glutamine adjacent to the flavin chromophore. The reaction cascade, triggered by the initial photoexcitation of the flavin chromophore, involves isomerization of this residue but no rotation as assumed previously. The fact that only the environment, but not the flavin chromophore by itself, is chemically transformed along the individual steps of the mechanism is unique for biological photoreceptors. The final isomer of the glutamine tautomer, i.e., the imidic acid, is further stabilized by the interchange of a methionine residue in the binding pocket with a tryptophan residue. The flip of these two residues might be the trigger for the large conformational change of this protein which is consequently transmitted as the signal to the biological environment

Theoretical investigations of the hydrogen bond in a tetraamido/diamino quaternized macrocycle

We present theoretical investigations on the nature and persistence of the strong central hydrogen bond of a tetraamido/diamino quaternized macrocycle. Our theoretical study of the NMR properties of the central hydrogen bond proved difficult to reconcile with the available experimental results, suggesting the possibility of the non-persistence of the central hydrogen bond of the macrocycle in solution. We demonstrate alternative scenarios, in which a tautomerization of the macrocycle in solution gives rise to the experimentally observed NMR shifts.</p

A Dynamic Equilibrium of Three Hydrogen-Bond Conformers Explains the NMR Spectrum of the Active Site of Photoactive Yellow Protein

A theoretical study on the NMR shifts of the hydrogen bond network around the chromophore, para-coumaric acid (<i>p</i>CA), of photoactive yellow protein (PYP) is presented. Previous discrepancies between theoretical and experimental studies are resolved by our findings of a previously unknown rapid conformational exchange near the active site of PYP. This exchange caused by the rotation of Thr50 takes place in the ground state of PYP’s active site and results in three effectively energetically equal conformations characterized by the formation of new hydrogen bonds, all of which contribute to the overall NMR signals of the investigated protons. In light of these findings, we are able to successfully explain the experimental results and provide valuable insight into the behavior of PYP in solution. We further investigated related PYP mutants (T50V, E46Q, and Y42F), and found the same conformational exchange in E46Q and Y42F to be responsible for the experimentally observed NMR and UV/vis spectra

Theoretical Study on the Repair Mechanism of the (6−4) Photolesion by the (6−4) Photolyase

UV irradiation of DNA can lead to the formation of mutagenic (6−4) pyrimidine−pyrimidone photolesions. The (6−4) photolyases are the enzymes responsible for the photoinduced repair of such lesions. On the basis of the recently published crystal structure of the (6−4) photolyase bound to DNA [Maul et al. 2008] and employing quantum mechanics/molecular mechanics techniques, a repair mechanism is proposed, which involves two photoexcitations. The flavin chromophore, initially being in its reduced anionic form, is photoexcited and donates an electron to the (6−4) form of the photolesion. The photolesion is then protonated by the neighboring histidine residue and forms a radical intermediate. The latter undergoes a series of energy stabilizing hydrogen-bonding rearrangements before the electron back transfer to the flavin semiquinone. The resulting structure corresponds to the oxetane intermediate, long thought to be formed upon DNA−enzyme binding. A second photoexcitation of the flavin promotes another electron transfer to the oxetane. Proton donation from the same histidine residue allows for the splitting of the four-membered ring, hence opening an efficient pathway to the final repaired form. The repair of the lesion by a single photoexcitation was shown not to be feasible

Effect of Including Torsional Parameters for Histidine–Metal Interactions in Classical Force Fields for Metalloproteins

Classical force-field parameters of the metal site of metalloproteins usually comprise only the partial charges of the involved atoms, as well as the bond-stretching and bending parameters of the metal–ligand interactions. Although for certain metal ligands such as histidine residues, the torsional motions at the metal site play an important role for the dynamics of the protein, no such terms have been considered to be crucial in the parametrization of the force fields, and they have therefore been omitted in the parametrization. In this work, we have optimized AMBER-compatible force-field parameters for the reduced state of the metal site of copper, zinc superoxide dismutase (SOD1) and assessed the effect of including torsional parameters for the histidine–metal interactions in molecular dynamics simulations. On the basis of the obtained results, we recommend that torsion parameters of the metal site are included when processes at the metal site are investigated or when free-energy calculations are performed. As the torsion parameters mainly affect the structure of the metal site, other kinds of structural studies can be performed without considering the torsional parameters of the metal site

Deamination, Oxidation, and C–C Bond Cleavage Reactivity of 5‑Hydroxymethylcytosine, 5‑Formylcytosine, and 5‑Carboxycytosine

Three new cytosine derived DNA modifications, 5-hydroxymethyl-2′-deoxycytidine (hmdC), 5-formyl-2′-deoxycytidine (fdC) and 5-carboxy-2′-deoxycytidine (cadC) were recently discovered in mammalian DNA, particularly in stem cell DNA. Their function is currently not clear, but it is assumed that in stem cells they might be intermediates of an active demethylation process. This process may involve base excision repair, C–C bond cleaving reactions or deamination of hmdC to 5-hydroxymethyl-2′-deoxyuridine (hmdU). Here we report chemical studies that enlighten the chemical reactivity of the new cytosine nucleobases. We investigated their sensitivity toward oxidation and deamination and we studied the C–C bond cleaving reactivity of hmdC, fdC, and cadC in the absence and presence of thiols as biologically relevant (organo)­catalysts. We show that hmdC is in comparison to mdC rapidly oxidized to fdC already in the presence of air. In contrast, deamination reactions were found to occur only to a minor extent. The C–C bond cleavage reactions require the presence of high concentration of thiols and are acid catalyzed. While hmdC dehydroxymethylates very slowly, fdC and especially cadC react considerably faster to dC. Thiols are active site residues in many DNA modifiying enzymes indicating that such enzymes could play a role in an alternative active DNA demethylation mechanism via deformylation of fdC or decarboxylation of cadC. Quantum-chemical calculations support the catalytic influence of a thiol on the C–C bond cleavage