Mismatch repair is a highly conserved cellular pathway responsible for repairing mismatched dsDNA. Errors are detected by the MutS enzyme, which most likely senses altered mechanical property of damaged dsDNA rather than a specific molecular pattern. While the curved shape of dsDNA in crystallographic MutS/DNA structures suggests the role of DNA bending, the theoretical support is not fully convincing. Here, we present a computational study focused on a base-pair opening into the minor groove, a specific base-pair motion observed upon interaction with MutS. Propensities for the opening were evaluated in terms of two base-pair parameters: Opening and Shear. We tested all possible base pairs in anti/anti, anti/syn and syn/anti orientations and found clear discrimination between mismatches and canonical base-pairs only for the opening into the minor groove. Besides, the discrimination gap was also confirmed in hotspot and coldspot sequences, indicating that the opening could play a more significant role in the mismatch recognition than previously recognized. Our findings can be helpful for a better understanding of sequence-dependent mutability. Further, detailed structural characterization of mismatches can serve for designing anti-cancer drugs targeting mismatched base pairs.
- MeSH
- chybné párování bází * MeSH
- DNA chemie metabolismus MeSH
- oprava chybného párování bází DNA * MeSH
- párování bází MeSH
- simulace molekulární dynamiky * MeSH
- termodynamika MeSH
- vazebný protein MutS opravné syntézy DNA chemie genetika metabolismus MeSH
- vodíková vazba MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mutations can be induced by environmental factors but also arise spontaneously during DNA replication or due to deamination of methylated cytosines at CpG dinucleotides. Sites where mutations occur with higher frequency than would be expected by chance are termed hotspots while sites that contain mutations rarely are termed coldspots. Mutations are permanently scanned and repaired by repair systems. Among them, the mismatch repair targets base pair mismatches, which are discriminated from canonical base pairs by probing altered elasticity of DNA. Using biased molecular dynamics simulations, we investigated the elasticity of coldspots and hotspots motifs detected in human genes associated with inherited disorders, and also of motifs with Czech population hotspots and de novo mutations. Main attention was paid to mutations leading to G/T and A+/C pairs. We observed that hotspots without CpG/CpHpG sequences are less flexible than coldspots, which indicates that flexible sequences are more effectively repaired. In contrary, hotspots with CpG/CpHpG sequences exhibited increased flexibility as coldspots. Their mutability is more likely related to spontaneous deamination of methylated cytosines leading to C > T mutations, which are primarily targeted by base excision repair. We corroborated conclusions based on computer simulations by measuring melting curves of hotspots and coldspots containing G/T mismatch.
- MeSH
- CpG ostrůvky MeSH
- DNA chemie genetika MeSH
- lidé MeSH
- mutace * MeSH
- nukleotidové motivy * MeSH
- simulace molekulární dynamiky * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BsoBI is a type II restriction endonuclease belonging to the EcoRI family. There is only one previously published X-ray structure for this endonuclease: it shows a homodimer of BsoBI completely encircling DNA in a tunnel. In this work, molecular dynamics simulations were employed to elucidate possible ways in which DNA is loaded into this complex prior to its cleavage. We found that the dimer does not open spontaneously when DNA is removed from the complex on the timescale of our simulations (~ 0.5 μs). A biased simulation had to be used to facilitate the opening, which revealed a possible way for the two catalytic domains to separate. The α-helices connecting the catalytic and helical domains were found to act as a hinge during the separation. In addition, we found that the opening of the BsoBI dimer was influenced by the type of counterions present in the environment. A reference simulation of the BsoBI/DNA complex further showed spontaneous reorganization of the active sites due to the binding of solvent ions, which led to an active-site structure consistent with other experimental structures of type II restriction endonucleases determined in the presence of metal ions.
Mutations in human genes can be responsible for inherited genetic disorders and cancer. Mutations can arise due to environmental factors or spontaneously. It has been shown that certain DNA sequences are more prone to mutate. These sites are termed hotspots and exhibit a higher mutation frequency than expected by chance. In contrast, DNA sequences with lower mutation frequencies than expected by chance are termed coldspots. Mutation hotspots are usually derived from a mutation spectrum, which reflects particular population where an effect of a common ancestor plays a role. To detect coldspots/hotspots unaffected by population bias, we analysed the presence of germline mutations obtained from HGMD database in the 5-nucleotide segments repeatedly occurring in genes associated with common inherited disorders, in particular, the PAH, LDLR, CFTR, F8, and F9 genes. Statistically significant sequences (mutational motifs) rarely associated with mutations (coldspots) and frequently associated with mutations (hotspots) exhibited characteristic sequence patterns, e.g. coldspots contained purine tract while hotspots showed alternating purine-pyrimidine bases, often with the presence of CpG dinucleotide. Using molecular dynamics simulations and free energy calculations, we analysed the global bending properties of two selected coldspots and two hotspots with a G/T mismatch. We observed that the coldspots were inherently more flexible than the hotspots. We assume that this property might be critical for effective mismatch repair as DNA with a mutation recognized by MutSα protein is noticeably bent.
- MeSH
- DNA chemie genetika MeSH
- faktor VIII genetika MeSH
- genetická predispozice k nemoci MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- molekulární modely MeSH
- nukleotidové motivy MeSH
- protein CFTR genetika MeSH
- receptory LDL genetika MeSH
- simulace molekulární dynamiky MeSH
- zárodečné mutace * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
We studied sequence-dependent retention properties of synthetic 5'-terminal phosphate absent trinucleotides containing adenine, guanine and thymine through reversed-phase liquid chromatography (RPLC) and QSRR modelling. We investigated the influence of separation conditions, namely mobile phase composition (ion interaction agent content, pH and organic constituent content), on sequence-dependent separation by means of ion-interaction RPLC (II-RPLC) using two types of models: experimental design-artificial neural networks (ED-ANN), and linear regression based on molecular dynamics data. The aim was to determine those properties of the above-mentioned analytes responsible for the retention dependence of the sequence. Our results show that there is a deterministic relation between sequence and II-RPLC retention properties of the studied trinucleotides. Further, we can conclude that the higher the content of ion-interaction agent in the mobile phase, the more prominent these properties are. We also show that if we approximate the polar component of solvation energy in QSRR by the electrostatic work in transferring molecules from vacuum to water, and the non-polar component by the solvent accessible surface area, these parameters best describe the retention properties of trinucleotides. There are some exceptions to this finding, namely sequences 5'-NAN-3', 5'-ANN-3', 5'-TGN-3', 5'-NTA-3'and 5'-NGA-3' (N stands for generic nucleotide). Their role is still unknown, but since linear regression including these specific constellations showed a higher observable variance coverage than the model with only the basic descriptors, we may assume that solvent-analyte interactions are responsible for the exceptional behaviour of 5'-NAN-3' & 5'-ANN-3' trinucleotides and some intramolecular interactions of neighbouring nucleobases for 5'-TGN-3', 5'-NTA-3'and 5'-NGA-3' trinucleotides.
- MeSH
- adenin analogy a deriváty izolace a purifikace MeSH
- chromatografie s reverzní fází MeSH
- guanin analogy a deriváty izolace a purifikace MeSH
- kvantitativní vztahy mezi strukturou a aktivitou MeSH
- neuronové sítě (počítačové) MeSH
- oligonukleotidy izolace a purifikace MeSH
- rozpouštědla MeSH
- simulace molekulární dynamiky MeSH
- statická elektřina MeSH
- thymin analogy a deriváty izolace a purifikace MeSH
- voda MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
Hyperphenylalaninemia (HPA) is one of the most common metabolic disorders. HPA, which is transmitted by an autosomal recessive mode of inheritance, is caused by mutations of the phenylalanine hydroxylase gene. Most mutations are missense and lead to reduced protein stability and/or impaired catalytic function. The impact of such mutations varies, ranging from classical phenylketonuria (PKU), mild PKU, to non-PKU HPA phenotypes. Despite the fact that HPA is a monogenic disease, clinical data show that one PKU genotype can be associated with more in vivo phenotypes, which indicates the role of other (still unknown) factors. To better understand the phenotype-genotype relationships, we analyzed computationally the impact of missense mutations in homozygotes stored in the BIOPKU database. A total of 34 selected homozygous genotypes was divided into two main groups according to their phenotypes: (A) genotypes leading to non-PKU HPA or combined phenotype non-PKU HPA/mild PKU and (B) genotypes leading to classical PKU, mild PKU or combined phenotype mild PKU/classical PKU. Combining in silico analysis and molecular dynamics simulations (in total 3 μs) we described the structural impact of the mutations, which allowed us to separate 32 out of 34 mutations between groups A and B. Testing the simulation conditions revealed that the outcome of mutant simulations can be modulated by the ionic strength. We also employed programs SNPs3D, Polyphen-2, and SIFT but based on the predictions performed we were not able to discriminate mutations with mild and severe PKU phenotypes.
- MeSH
- fenotyp MeSH
- fenylalaninhydroxylasa chemie genetika MeSH
- fenylketonurie genetika patologie MeSH
- genotyp MeSH
- konformace proteinů MeSH
- lidé MeSH
- missense mutace genetika MeSH
- počítačová simulace MeSH
- simulace molekulární dynamiky MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The isotropic (129)Xe nuclear magnetic resonance (NMR) chemical shift (CS) in Xe@C60 dissolved in liquid benzene was calculated by piecewise approximation to faithfully simulate the experimental conditions and to evaluate the role of different physical factors influencing the (129)Xe NMR CS. The (129)Xe shielding constant was obtained by averaging the (129)Xe nuclear magnetic shieldings calculated for snapshots obtained from the molecular dynamics trajectory of the Xe@C60 system embedded in a periodic box of benzene molecules. Relativistic corrections were added at the Breit-Pauli perturbation theory (BPPT) level, included the solvent, and were dynamically averaged. It is demonstrated that the contribution of internal dynamics of the Xe@C60 system represents about 8% of the total nonrelativistic NMR CS, whereas the effects of dynamical solvent add another 8%. The dynamically averaged relativistic effects contribute by 9% to the total calculated (129)Xe NMR CS. The final theoretical value of 172.7 ppm corresponds well to the experimental (129)Xe CS of 179.2 ppm and lies within the estimated errors of the model. The presented computational protocol serves as a prototype for calculations of (129)Xe NMR parameters in different Xe atom guest-host systems.
Vectorial proton transfer among carbonyl oxygen atoms was studied in two models of tripeptide via quantum chemical calculations using the hybrid B3LYP functional and the 6-31++G basis set. Two principal proton transfer pathways were found: a first path involving isomerization of the proton around the double bond of the carbonyl group, and a second based on the large conformational flexibility of the tripeptide model where all carbonyl oxygen atoms cooperate. The latter pathway has a rate-determining step energy barrier that is only around half of that for the first pathway. As conformational flexibility plays a crucial role in second pathway, the effect of attaching methyl groups to the alpha carbon atoms was studied. The results obtained are presented for all four possible stereochemical configurations.
