Metadynamics simulation study on the conformational transformation of HhaI methyltransferase: an induced-fit base-flipping hypothesis.
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ABSTRACT:
SUBMITTER: Jin L
PROVIDER: S-EPMC4090504 | biostudies-literature | 2014
REPOSITORIES: biostudies-literature
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Project description:Rotation of a DNA or RNA nucleotide out of the double helix and into a protein pocket ('base flipping') is a mechanistic feature common to some DNA/RNA-binding proteins. Here, we report the structure of HhaI methyltransferase in complex with DNA containing a south-constrained abasic carbocyclic sugar at the target site in the presence of the methyl donor byproduct AdoHcy. Unexpectedly, the locked south pseudosugar appears to be trapped in the middle of the flipping pathway via the DNA major groove, held in place primarily through Van der Waals contacts with a set of invariant amino acids. Molecular dynamics simulations indicate that the structural stabilization observed with the south-constrained pseudosugar will not occur with a north-constrained pseudosugar, which explains its lowered binding affinity. Moreover, comparison of structural transitions of the sugar and phosphodiester backbone observed during computational studies of base flipping in the M.HhaI-DNA-AdoHcy ternary complex indicate that the south-constrained pseudosugar induces a conformation on the phosphodiester backbone that corresponds to that of a discrete intermediate of the base-flipping pathway. As previous crystal structures of M.HhaI ternary complex with DNA displayed the flipped sugar moiety in the antipodal north conformation, we suggest that conversion of the sugar pucker from south to north beyond the middle of the pathway is an essential part of the mechanism through which flipping must proceed to reach its final destination. We also discuss the possibility of the south-constrained pseudosugar mimicking a transition state in the phosphodiester and sugar moieties that occurs during DNA base flipping in the presence of M.HhaI.
| S-EPMC506793 | biostudies-literature
Project description:Individual DNA bases are known to be able to flip out of the helical stack, providing enzymes with access to the genetic information otherwise hidden inside the helix. Consequently, base flipping is a necessary first step to many more complex biological processes such as DNA transcription or replication. Much remains unknown about this elementary step, despite a wealth of experimental and theoretical studies. From the theoretical point of view, the involved timescale of milliseconds or longer requires the use of enhanced sampling techniques. In contrast to previous theoretical studies employing umbrella sampling along a predefined flipping coordinate, this study attempts to induce flipping without prior knowledge of the pathway, using information from a molecular dynamics simulation of a B-DNA fragment and the conformational flooding method. The relevance to base flipping of the principal components of the simulation is assayed, and a combination of modes optimally related to the flipping of the base through either helical groove is derived for each of the two bases of the central guanine-cytosine basepair. By applying an artificial flooding potential along these collective coordinates, the flipping mechanism is accelerated to within the scope of molecular dynamics simulations. The associated free energy surface is found to feature local minima corresponding to partially flipped states, particularly relevant to flipping in isolated DNA; further transitions from these minima to the fully flipped conformation are accelerated by additional flooding potentials. The associated free energy profiles feature similar barrier heights for both bases and pathways; the flipped state beyond is a broad and rugged attraction basin, only a few kcal/mol higher in energy than the closed conformation. This result diverges from previous works but echoes some aspects of recent experimental findings, justifying the need for novel approaches to this difficult problem: this contribution represents a first step in this direction. Important structural factors involved in flipping, both local (sugar-phosphate backbone dihedral angles) and global (helical axis bend), are also identified.
| S-EPMC1913169 | biostudies-literature
Project description:The U6 RNA internal stem-loop (U6 ISL) is a highly conserved domain of the spliceosome that is important for pre-mRNA splicing. The U6 ISL contains an internal loop that is in equilibrium between two conformations controlled by the protonation state of an adenine (pK(a)=6.5). Lower pH favors formation of a protonated C-A(+) wobble pair and base flipping of the adjacent uracil. Higher pH favors stacking of the uracil and allows an essential metal ion to bind at this position. Here, we define the minimal-energy path for this conformational transition. To do this, we solved the U6 ISL structure at higher pH (8.0) in order to eliminate interference from the low-pH conformer. This structure reveals disruption of the protonated C-A(+) pair and formation of a new C-U pair, which explains the preference for a stacked uracil at higher pH. Next, we used nudged elastic band molecular dynamics simulations to calculate the minimum-energy path between the two conformations. Our results indicate that the C-U pair is dynamic, which allows formation of the more stable C-A(+) pair upon adenine protonation. After formation of the C-A(+) pair, the unpaired uracil follows a minor-groove base-flipping pathway. Molecular dynamics simulations suggest that the extrahelical uracil is stabilized by contacts with the adjacent helix.
| S-EPMC2799254 | biostudies-literature
Project description:DNA base modifications and mutations are observed in all genomes throughout the kingdoms of life. Proteins involved in their establishment and removal were shown to use a base flipping mechanism to access their substrates. To better understand how proteins flip DNA bases to modify or remove them, we optimized and developed a pipeline of methods to step-by-step detect the process starting with protein-DNA interaction, base flipping itself and the ensuing DNA base modification or excision. As methylcytosine is the best-studied DNA modification, here we focus on the process of writing, modifying and reading this DNA base. Using multicolor electrophoretic mobility shift assays, we show that the methylcytosine modifier Tet1 exhibits little DNA sequence specificity with only a slight preference for methylated CpG containing DNA. A combination of chloroacetaldehyde treatment and high-resolution melting temperature analysis allowed us to detect base flipping induced by the methylcytosine modifier Tet1 as well as the methylcytosine writer M.HpaII. Finally, we show that high-resolution melting temperature analysis can be used to detect the activity of glycosylases, methyltransferases and dioxigenases on DNA substrates. Taken together, this DNA base flipping analytical pipeline (BaFAP) provide a complete toolbox for the fast and sensitive analysis of proteins that bind, flip and modify or excise DNA bases.
| S-EPMC6994035 | biostudies-literature
Project description:On the basis of non-covalent bond interactions in nucleic acids, we synthesized the deoxyadenosine derivatives tethering a phenyl group (X) and a naphthyl group (Z) by an amide linker, which mimic a Watson-Crick base pair. Circular dichroism spectra indicated that the duplexes containing X and Z formed a similar conformation regardless of the opposite nucleotide species (A, G, C, T and an abasic site analogue F), which was not observed for the natural duplexes. The values among the natural duplexes containing the A/A, A/G, A/C, A/T and A/F pairs differed by 5.2 kcal mol(-1) while that among the duplexes containing X or Z in place of the adenine differed by only 1.9 or 2.8 kcal mol(-1), respectively. Fluorescence quenching experiments confirmed that 2-amino purine opposite X adopted an unstacked conformation. The structural and thermodynamic analyses suggest that the aromatic hydrocarbon group of X and Z intercalates into a double helix, resulting in the opposite nucleotide base flipping into an unstacked position regardless of the nucleotide species. This observation implies that modifications at the aromatic hydrocarbon group and the amide linker may expand the application of the base pair-mimic nucleosides for molecular biology and biotechnology.
| S-EPMC1316115 | biostudies-literature
Project description:We have analysed the DNA-protein contacts made between the type I DNA methyltransferase M.EcoR124I and its recognition sequence. The effects of base modifications have been probed by measuring the affinity of M.EcoR124I for the modified sequences relative to that for the wild-type sequence by using gel-retardation competition assays. These results, along with those from methylation interference footprinting and photo-affinity cross-linking have identified the location of potential DNA contacts within the DNA recognition site. Substitution of 6-thioguanosine for each of the three specific guanines in the recognition sequence leads to a large (10-20-fold) decrease in the strength of DNA binding, indicating the importance of hydrogen-bonding interactions in the major groove of DNA. In contrast, replacement of either (or both) of the adenines at the target site for methylation by the enzyme, to produce either a base pair mismatch or loss of the base, leads to a marked increase in DNA-binding affinity. The results strongly support the proposal that type I methyltransferases employ a base-flipping mechanism to methylate their target base.
| S-EPMC1219925 | biostudies-other
Project description:The genome of Mycoplasma arthritidis strain 158 has modified cytosine residues at AGCT sequences that render the DNA resistant to digestion with the AluI restriction endonuclease. The DNA methyltransferase responsible for the base modification has previously been designated MarI. From the complete genome sequence of M. arthritidis, we identify Marth_orf138 as a candidate marI gene. Marth_orf138 was cloned in Escherichia coli and its TGA codons converted to TGG. DNA isolated from E. coli cells expressing the modified Marth_orf138 gene was degraded by the AluI nuclease, indicating that Marth_orf138 does not code for MarI. However, the DNA from E. coli was found to have acquired resistance to the restriction endonuclease HhaI. Genomic DNA from M. arthritidis was also found to be resistant to HhaI (recognizes GCGC). The M. arthritidis isoschizomer of the HhaI DNA methyltransferase, coded by Marth_orf138, is designated MarII. Transformation of M. arthritidis was not significantly affected by modification of plasmid at HhaI sites, indicating that the mycoplasma lacks a restriction endonuclease that recognizes GCGC sites.
| S-EPMC2668981 | biostudies-literature
Project description:The sequence-dependent structural variability and conformational dynamics of DNA play pivotal roles in many biological milieus, such as in the site-specific binding of transcription factors to target regulatory elements. To better understand DNA structure, function, and dynamics in general, and protein...DNA recognition in the 'kappaB' family of genetic regulatory elements in particular, we performed molecular dynamics simulations of a 20-bp DNA encompassing a cognate kappaB site recognized by the proto-oncogenic 'c-Rel' subfamily of NF-kappaB transcription factors. Simulations of the kappaB DNA in explicit water were extended to microsecond duration, providing a broad, atomically detailed glimpse into the structural and dynamical behavior of double helical DNA over many timescales. Of particular note, novel (and structurally plausible) conformations of DNA developed only at the long times sampled in this simulation-including a peculiar state arising at approximately 0.7 micros and characterized by cross-strand intercalative stacking of nucleotides within a longitudinally sheared base pair, followed (at approximately 1 micros) by spontaneous base flipping of a neighboring thymine within the A-rich duplex. Results and predictions from the microsecond-scale simulation include implications for a dynamical NF-kappaB recognition motif, and are amenable to testing and further exploration via specific experimental approaches that are suggested herein.
| S-EPMC2528173 | biostudies-literature
Project description:DNA mismatch recognition and repair is vital for preserving the fidelity of the genome. Conserved across prokaryotes and eukaryotes, MutS is the primary protein that is responsible for recognizing a variety of DNA mismatches. From molecular dynamics simulations of the Escherichia coli MutS-DNA complex, we describe significant conformational dynamics in the DNA surrounding a G·T mismatch that involves weakening of the basepair hydrogen bonding in the basepair adjacent to the mismatch and, in one simulation, complete base opening via the major groove. The energetics of base flipping was further examined with Hamiltonian replica exchange free energy calculations revealing a stable flipped-out state with an initial barrier of ~2 kcal/mol. Furthermore, we observe changes in the local DNA structure as well as in the MutS structure that appear to be correlated with base flipping. Our results suggest a role of base flipping as part of the repair initiation mechanism most likely leading to sliding-clamp formation.
| S-EPMC3207177 | biostudies-literature
Project description:PspGI is a representative of a group of restriction endonucleases that recognize a pentameric sequence related to CCNGG. Unlike the previously investigated Ecl18kI, which does not have any specificity for the central base pair, PspGI prefers A/T over G/C in its target site. Here, we present a structure of PspGI with target DNA at 1.7 A resolution. In this structure, the bases at the center of the recognition sequence are extruded from the DNA and flipped into pockets of PspGI. The flipped thymine is in the usual anti conformation, but the flipped adenine takes the normally unfavorable syn conformation. The results of this and the accompanying manuscript attribute the preference for A/T pairs over G/C pairs in the flipping position to the intrinsically lower penalty for flipping A/T pairs and to selection of the PspGI pockets against guanine and cytosine. Our data show that flipping can contribute to the discrimination between normal bases. This adds a new role to base flipping in addition to its well-known function in base modification and DNA damage repair.
| S-EPMC2577326 | biostudies-literature