DNA binding properties in vivo and target recognition domain sequence alignment analyses of wild-type and mutant RsrI [N6-adenine] DNA methyltransferases

A genetic selection method, the P22 challenge-phage assay, was used to characterize DNA binding in vivo by the prokaryotic beta class [N:6-adenine] DNA methyltransferase M.RSR:I. M.RSR:I mutants with altered binding affinities in vivo were isolated. Unlike the wild-type enzyme, a catalytically compromised mutant, M.RSR:I (L72P), demonstrated site-specific DNA binding in vivo. The L72P mutation is located near the highly conserved catalytic motif IV, DPPY (residues 65-68). A double mutant, M.RSR:I (L72P/D173A), showed less binding in vivo than did M.RSR:I (L72P). Thus, introduction of the D173A mutation deleteriously affected DNA binding. D173 is located in the putative target recognition domain (TRD) of the enzyme. Sequence alignment analyses of several beta class MTases revealed a TRD sequence element that contains the D173 residue. Phylogenetic analysis suggested that divergence in the amino acid sequences of these methyltransferases correlated with differences in their DNA target recognition sequences. Furthermore, MTases of other classes (alpha and gamma) having the same DNA recognition sequence as the beta class MTases share related regions of amino acid sequences in their TRDs.     

Structure of RsrI methyltransferase, a member of the N6-adenine beta class of DNA methyltransferases

DNA methylation is important in cellular, developmental and disease processes, as well as in bacterial restriction-modification systems. Methylation of DNA at the amino groups of cytosine and adenine is a common mode of protection against restriction endonucleases afforded by the bacterial methyltransferases. The first structure of an N:6-adenine methyltransferase belonging to the beta class of bacterial methyltransferases is described here. The structure of M. RSR:I from Rhodobacter sphaeroides, which methylates the second adenine of the GAATTC sequence, was determined to 1.75 A resolution using X-ray crystallography. Like other methyltransferases, the enzyme contains the methylase fold and has well-defined substrate binding pockets. The catalytic core most closely resembles the PVU:II methyltransferase, a cytosine amino methyltransferase of the same beta group. The larger nucleotide binding pocket observed in M. RSR:I is expected because it methylates adenine. However, the most striking difference between the RSR:I methyltransferase and the other bacterial enzymes is the structure of the putative DNA target recognition domain, which is formed in part by two helices on an extended arm of the protein on the face of the enzyme opposite the active site. This observation suggests that a dramatic conformational change or oligomerization may take place during DNA binding and methylation.     

Specific recognition of DNA by integration host factor. Glutamic acid 44 of the beta-subunit specifies the discrimination of a T:A from an A:T base pair without directly contacting the DNA

Integration host factor (IHF) is a protein that binds to the H' site of bacteriophage lambda with sequence specificity. Genetic experiments implicated amino acid residue Glu(44) of the beta-subunit of IHF in discrimination against substitution of A for T at position 44 of the TTR submotif of the binding site (Lee, E. C., Hales, L. M., Gumport, R. I., Gardner, J. F. (1992) EMBO J., 11, 305-313). We have extended this observation by generating all possible single-base substitutions at positions 43, 44, and 45 of the H' site. IHF failed to bind these H' site substitution mutants in vivo. The K(d)(app) value for each H' site substitution, except for H'45A mutant, was reduced >2000-fold relative to the wild-type site. Substitution of amino acid beta-Glu(44) with alanine prevented IHF from discriminating against the H'44A variant but not the other H' site substitution mutants. Further analysis with other substitutions at position beta44 demonstrated that both oxygens of the wild-type glutamic acid are necessary for discrimination of AT at position 44. Because the beta-Glu(44) residue does not contact the DNA, this residue probably enforces binding specificity indirectly through interaction with amino acids that themselves contact the DNA.     

A new affinity reagent for the site-specific, covalent attachment of DNA to active-site nucleophiles: application to the EcoRI and RsrI restriction and modification enzymes.

A modified oligodeoxyribonucleotide duplex containing an unnatural internucleotide trisubstituted 3' to 5' pyrophosphate bond in one strand [5'(oligo1)3'-P(OCH3)P-5'(oligo2) 3'] reacts with nucleophiles in aqueous media by acting as a phosphorylating affinity reagent. When interacted with a protein, a portion of the oligonucleotide [--P-5'(oligo2)3'] becomes attached to an amino acid nucleophilic group through a phosphate of the O-methyl-modified pyrophosphate linkage. We demonstrate the affinity labeling of nucleophilic groups at the active sites of the EcoRI and RsrI restriction and modification enzymes with an oligodeoxyribonucleotide duplex containing a modified scissile bond in the EcoRI recognition site. With the EcoRI and RsrI endonucleases in molar excess approximately 1% of the oligonucleotide becomes attached to the protein, and with the companion methyltransferases the yield approaches 40% for the EcoRI enzyme and 30% for the RsrI methyltransferase. Crosslinking proceeds only upon formation of a sequence-specific enzyme-DNA complex, and generates a covalent bond between the 3'-phosphate of the modified pyrophosphate in the substrate and a nucleophilic group at the active site of the enzyme. The reaction results in the elimination of an oligodeoxyribonucleotide remnant that contains the 3'-O-methylphosphate [5'(oligo1)3'-P(OCH3)] derived from the modified phosphate of the pyrophosphate linkage. Hydrolysis properties of the covalent protein-DNA adducts indicate that phosphoamide (P-N) bonds are formed with the EcoRI endonuclease and methyltransferase.     

A genetic enrichment for mutations constructed by oligodeoxynucleotide-directed mutagenesis

A genetic enrichment procedure for mutations constructed by oligodeoxynucleotide(oligo)-directed mutagenesis of DNA cloned in M13mp vectors is described. The procedure uses an M13 vector that contains the cloned target DNA and amber (am) mutations within the phage genes I and II. This vector cannot replicate in a suppressor-free (sup degrees) bacterial strain. A gapped heteroduplex is formed by annealing portions of a complementary (-)strand containing wild-type copies of genes I and II to the am-containing template (+)strand. The oligo is annealed to the single-stranded (ss) region and the remaining gaps and nicks are repaired enzymatically to form a closed circular heteroduplex structure. By transfecting the DNA into a sup degrees host we promote the propagation of heteroduplexes with the oligo-containing (-)strand since only this construction contains the wild-type copies of genes I and II. This procedure eliminates the need for any physical separation of the covalently closed circular DNA that contains the oligo from the ss template. Using this technique we have constructed 17 point mutations with mutation frequencies ranging from 2-20% for single base changes and from 0.3-9% for multiple base changes. In addition, we found that the mutation frequencies were affected by the state of DNA methylation in the (+) and (-)strands.     

Adult spinal opioid receptor μ1 expression after incision is altered by early life repetitive tactile and noxious procedures in rats

Clinical and experimental data suggests that noxious stimulation at critical stages of development results in long‐term changes on nociceptive processing in later life. Here, we use an established, well‐documented rat model of repetitive noxious procedures closely mimicking the clinical situation in the NICU. In order to understand molecular changes underlying the long‐term consequences of repetitive stimulation of the developing nociceptive system the present study aims to analyze the presence of the µ‐opioid‐receptor‐1 (OPRM1). Neonatal rats received either four needle pricks per day in the left hind‐paw from postnatal day 0–7 as a model of procedural pain in infancy. Control pups were handled in the same way but were instead tactile stimulated, or were left undisturbed. At the age of 8 weeks, all animals received an ipsilateral hind‐paw incision as a model for post‐operative pain, and mechanical sensitivity was tested at multiple time‐points. Before, and 1 or 5 days post‐incision, spinal cord tissue was collected for immunostaining of opioid receptor OPRM1. Semi‐quantitative immunocytochemical analysis of superficial laminae in lumbar spinal dorsal horn revealed that: (1) early life repetitive tactile or noxious procedures do not alter baseline levels of OPRM1 staining intensity and (2) early life repetitive tactile or noxious procedures lead to a decrease in OPRM1 staining intensity 5 days after incision in adulthood compared to undisturbed controls. We conclude that early life repetitive tactile or noxious procedures affect the intensity of OPRM1‐immunoreactivity in the lumbar superficial spinal cord dorsal horn after adulthood injury, without affecting baseline intensity. © 2018 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol 78: 417–426, 2018     

Dimerization of the bacterial RsrI N6-adenine DNA methyltransferase

Dimeric restriction endonucleases and monomeric modification methyltransferases were long accepted as the structural paradigm for Type II restriction systems. Recent studies, however, have revealed an increasing number of apparently dimeric DNA methyltransferases. Our initial characterization of RsrI methyltransferase (M.RsrI) was consistent with the enzyme functioning as a monomer, but, subsequently, the enzyme crystallized as a dimer with 1500 A2 of buried surface area. This result led us to re-examine the biochemical properties of M.RsrI. Gel-shift studies of M.RsrI binding to DNA suggested that binding cooperativity targets hemimethylated DNA preferentially over unmethylated DNA. Size-exclusion chromatography indicated that the M.RsrI-DNA complex had a size and stoichiometry consistent with a dimeric enzyme binding to the DNA. Kinetic measurements revealed a quadratic relationship between enzyme velocity and concentration. Site-directed mutagenesis at the dimer interface affected the kinetics and DNA-binding of the enzyme, providing support for a model proposing an active enzyme dimer. We also identified a conserved motif in the dimer interfaces of the beta-class methyltransferases M.RsrI, M.MboIIA and M2.DpnII. Taken together, these data suggest that M.RsrI may be part of a sub-class of MTases that function as dimers.     

Structures of liganded and unliganded RsrI N6-adenine DNA methyltransferase: a distinct orientation for active cofactor binding

The structures of RsrI DNA methyltransferase (M.RsrI) bound to the substrate S-adenosyl-l-methionine (AdoMet), the product S-adenosyl-l-homocysteine (AdoHcy), the inhibitor sinefungin, as well as a mutant apo-enzyme have been determined by x-ray crystallography. Two distinct binding configurations were observed for the three ligands. The substrate AdoMet adopts a bent shape that directs the activated methyl group toward the active site near the catalytic DPPY motif. The product AdoHcy and the competitive inhibitor sinefungin bind with a straight conformation in which the amino acid moiety occupies a position near the activated methyl group in the AdoMet complex. Analysis of ligand binding in comparison with other DNA methyltransferases reveals a small, common subset of available conformations for the ligand. The structures of M.RsrI with the non-substrate ligands contained a bound chloride ion in the AdoMet carboxylate-binding pocket, explaining its inhibition by chloride salts. The L72P mutant of M.RsrI is the first DNA methyltransferase structure without bound ligand. With respect to the wild-type protein, it had a larger ligand-binding pocket and displayed movement of a loop (223-227) that is responsible for binding the ligand, which may account for the weaker affinity of the L72P mutant for AdoMet. These studies show the subtle changes in the tight specific interactions of substrate, product, and an inhibitor with M.RsrI and help explain how each displays its unique effect on the activity of the enzyme.