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.

Substrate binding in vitro and kinetics of RsrI [N6-adenine] DNA methyltransferase.

RSR:I [N:6-adenine] DNA methyltransferase (M.RSR:I), which recognizes GAATTC and is a member of a restriction-modification system in Rhodobacter sphaeroides, was purified to >95% homogeneity using a simplified procedure involving two ion exchange chromatographic steps. Electrophoretic gel retardation assays with purified M.RSR:I were performed on unmethylated, hemimethylated, dimethylated or non-specific target DNA duplexes (25 bp) in the presence of sinefungin, a potent inhibitory analog of AdoMet. M. RSR:I binding was affected by the methylation status of the DNA substrate and was enhanced by the presence of the cofactor analog. M. RSR:I bound DNA substrates in the presence of sinefungin with decreasing affinities: hemimethylated > unmethylated > dimethylated >> non-specific DNA. Gel retardation studies with DNA substrates containing an abasic site substituted for the target adenine DNA provided evidence consistent with M.RSR:I extruding the target base from the duplex. Consistent with such base flipping, an approximately 1.7-fold fluorescence intensity increase was observed upon stoichiometric addition of M.RSR:I to hemimethylated DNA containing the fluorescent analog 2-aminopurine in place of the target adenine. Pre-steady-state kinetic and isotope- partitioning experiments revealed that the enzyme displays burst kinetics, confirmed the catalytic competence of the M.RSR:I-AdoMet complex and eliminated the possibility of an ordered mechanism where DNA is required to bind first. The equilibrium dissociation constants for AdoMet, AdoHcy and sinefungin were determined using an intrinsic tryptophan fluorescence-quenching assay.

Probing the role of structural water in a duplex oligodeoxyribonucleotide containing a water-mimicking base analog.

Molecular dynamics simulations were performed on models of the dodecamer DNA double-stranded segment, [d(CGCGAATTCGCG)](2), in which each of the adenine residues, individually or jointly, was replaced by the water-mimicking analog 2'-deoxy-7-(hydroxy-methyl)-7-deazaadenosine (hm(7)c(7)dA) [Rockhill, J.K., Wilson,S.R. and Gumport,R.I. (1996) J. Am. Chem. Soc.,118, 10065-10068]. The simulations, when compared with those of the dodecamer itself, show that incorporation of the analog affects neither the overall DNA structure nor its hydrogen-bonding and stacking interactions when it replaces a single individual base. Furthermore, the water molecules near the bases in the singly-substituted oligonucleotides are similarly unaffected. Double substitutions lead to differences in all the aforementioned parameters with respect to the reference sequence. The results suggest that the analog provides a good mimic of specific 'ordered' water molecules observed in contact with DNA itself and at the interface between protein and DNA in specific complexes.

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.

The purification of nuclease-free T4-RNA ligase.

RNA ligase has been highly purified in good yields from bacteriophage T4-infected Escherichia coli by a rapid and reproducible procedure. The enzyme is free of phosphomonoesterase and ribonuclease activities and is therefore suitable for the synthesis of oligoribonucleotides and for the labeling of the 3'-terminus of RNA. Greater than 90% of the protein in the enzyme preparation migrates as a single band on gradient polyacrylamide gels containing sodium dodecyl sulfate during electrophoresis. For use as a DNA synthesis reagent the enzyme may be reliably freed of deoxyribonuclease activity by an additional chromatographic procedure using a commercially avialable resin.

Defining the structural and functional roles of the carboxyl region of the bacteriophage lambda excisionase (Xis) protein.

The bacteriophage lambda excisionase (Xis) protein is required for excisive site-specific recombination. Xis is composed of 72 amino acids and binds cooperatively to two DNA sites (X1 and X2) that are arranged as direct repeats. Alternatively, Xis binds cooperatively with the host-encoded factor for inversion stimulation (FIS) protein at the X1 and F sites, respectively. Here we analyzed the effects of missense substitutions from codon 57 to the carboxyl end of the protein and nonsense mutations that truncate the protein at various positions from residues 60 to 69. We find that all of the mutant proteins promote excision to some extent and interact cooperatively with FIS. Some mutants have no detectible phenotype while others are altered in their abilities to promote excision or to interact cooperatively with integrase (Int). Computer modeling predicts that amino acids from residues 59 to 65 are in an alpha-helix conformation. Mutants with substitutions on one side of the helix at residues 57, 60, 63 and 64 as well as truncated mutants containing 60, 61 or 63 amino acids, fail to interact cooperatively with Int suggesting that this region of the protein forms the interface with Int. Mutants with substitutions at other positions in the putative helix have no detectible phenotype. Residues 66 to 68 may form a reverse turn and the last four amino acids (69 to 72) may not be crucial for the structure or function of the protein.

Mapping the functional domains of bacteriophage lambda integrase protein.

Bacteriophage lambda encodes a site-specific recombination system that promotes the movement of the phage genome into and out of the host bacterial chromosome. The phage-encoded integrase (Int) is composed of 356 amino acid residues and carries out the required strand exchanges by means of a type I topoisomerase activity. Int also contains two distinct DNA-binding domains that interact with two different, specific sequences (arm-type and core-type sites) on DNA. In order to help understand the mechanism of site-specific recombination, we have used a genetic approach to isolate mutants defective in different steps in the recombination reaction. We developed a genetic screen for Int mutants that are defective in catalyzing excisive recombination in vivo. These mutants were screened for proficiency in binding to the P'123 arm-type sites using the bacteriophage P22 challenge-phage assays. In all, 78 such mutants were isolated and the mutational changes mapped and sequenced. These mutants have been further characterized (1) for their ability to bind the P'1 and P'123 arm-type sites and for their ability to form the attL complex in vivo, (2) for negative dominance in vitro, (3) for the presence of type I topoisomerase activity, and (4) for the ability to resolve artificially constructed recombination intermediates. We found that (1) residues in a stretch of 88 amino acids in the middle of the protein may be involved in Int-Int interactions, (2) a region around Arg212 is involved in the catalytic site, (3) residues near the carboxyl terminus play a role in enhancing Int binding to its arm-type sites, possibly by interacting with the small amino-terminal region that has been shown to be responsible for specific recognition of the arm-type sites, and (4) residues at the very carboxyl end of the protein may be involved in modulating the cleavage or religation activities of the Int protein.

Extent of sequence homology required for bacteriophage lambda site-specific recombination.

Bacteriophage lambda integration and excision occur by reciprocal recombination within a 15-base homologous core region present in the recombining attachment (att) sites. Strand exchange within the core occurs at precise nucleotide positions, which define an overlap region in which the products of recombination contain DNA strands derived from different parents. In order to define the role of sequence homology during recombination we have constructed point mutations within the core and assayed their effects in vivo and in vitro on site-specific recombination. Two of the mutations are located at position -3 of the core, which is one base-pair outside of the overlap region where strand exchange occurs. These mutations do not affect integrative or excisive recombination, thereby suggesting that homology outside the overlap region is not required for recombination. Two other mutations are located at position -2 of the core, which is one base-pair within the overlap region. These mutations show severely depressed integrative and excisive recombination activities in vitro and in vivo when recombined against wild-type att sites. However, the -2 mutations show normal recombination activity when recombined against att sites containing the homologous mutation, thereby suggesting that homology-dependent DNA interactions are required within the overlap region for effective recombination. In vitro recombination between homoduplex attP sites and heteroduplex attB sites demonstrated that the DNA interactions require only one strand of the attB overlap region to be homologous to attP in order to promote recombination.

Mutational analysis of integrase arm-type binding sites of bacteriophage lambda. Integration and excision involve distinct interactions of integrase with arm-type sites.

Integrative recombination between specific attachment (att) regions of the bacteriophage lambda genome (attP) and the Escherichia coli genome (attB) results in a prophage flanked by the hybrid recombinant sites attL and attR. Each att site contains sequences to which proteins involved in recombination bind. Using site-directed mutagenesis, we have constructed a related set of point mutations within each of the five Int "arm-type" binding sites located within attP, attL and attR. Footprint analyses of binding demonstrate that mutating the arm-type sites significantly disrupts the binding of Int. Recombination analyses of mutant att sites in vivo and in vitro demonstrate that only three wild-type arm-type sites within attP are required for efficient integrative recombination. Similar analyses demonstrate that efficient excision can occur with two other different sets of wild-type arm-type sites in attL and attR. These results demonstrate that integrative and excisive recombination may involve interactions of Int with distinct and different subsets of arm-type sites.

Specificity of the attenuation response of the threonine operon of Escherichia coli is determined by the threonine and isoleucine codons in the leader transcript.

Expression of the threonine (thr) operon enzymes of Escherichia coli is regulated by an attenuation mechanism. The regulatory portion of the operon contains a region coding for a leader peptide that contains consecutive threonine and isoleucine codons. It is thought that translation of the leader peptide controls the frequency of transcription termination at the attenuator site. Using oligonucleotide-directed site-specific mutagenesis we have altered the putative control codons of the leader peptide coding region. In two of the mutants the threonine and isoleucine codons were changed to produce peptides containing histidine and tyrosine codons. Both mutants showed loss of regulation by threonine and isoleucine. A hisT mutation, which leads to an undermodification of tRNA(His), increased thr operon expression in the mutants threefold but did not affect expression of the wild-type thr operon. Two other mutants were constructed that contained two histidine codons early in the leader peptide. Expression in both of these mutants was unaltered by the presence of the hisT allele or by the addition of threonine and isoleucine to the growth medium. In addition, a wild-type strain containing a temperature-sensitive threonyl-tRNA synthetase mutation showed increased thr operon expression at the non-permissive temperature, whereas none of the mutants showed any change. Taken together these data indicate that the specificity of the attenuation response is effected by specific control codons within the thr leader peptide coding region. We have also directly demonstrated thr leader peptide synthesis in vitro using a plasmid encoding the wild-type thr leader region to direct the synthesis of a peptide of the appropriate molecular weight when labeled with [3H]threonine but not with [3H]histidine or [3H]tyrosine. Conversely, when extracts were incubated with templates containing the mutated DNAs, peptides were labeled that showed patterns consistent with the expected amino acid compositions. These data indicate that the thr leader RNA is translated into the predicted leader peptide.

Genetic analysis of the bacteriophage lambda attL nucleoprotein complex.

Site-specific recombination in bacteriophage lambda involves interactions among proteins required for integration and excision of DNA molecules. We have analyzed the elements required to form an in vivo nucleoprotein complex of integrase (Int) and integration host factor (IHF). Interaction of Int with the core (the site of strand exchange) is stabilized by the flanking arm region of attL. IHF, in addition to Int, is required for efficient Int-core binding. We used the in vivo attL binding assay to characterize several Int variants for their abilities to form stable attL complexes. Substitution of Int active site tyrosine 342 by phenylalanine had no effect on the ability of the protein to form attL complexes. Three other amino acids that are completely conserved in the integrase family of recombinases (arginine 212, histidine 308, and arginine 311) were separately substituted by glutamine, leucine, and histidine, respectively. In each case, the mutant protein was altered in its ability to form attL complexes while retaining its ability to bind to the lambda arm-type sites. We propose that, in addition to their role in catalysis, this triad of amino acids helps the Int protein to interact with the lambda core sites.

The effect of attachment site mutations on strand exchange in bacteriophage lambda site-specific recombination.

Recombination of phage lambda attachment sites occurs by sequential exchange of the DNA strands at two specific locations. The first exchange produces a Holliday structure, and the second resolves it to recombinant products. Heterology for base substitution mutations in the region between the two strand exchange points (the overlap region) reduces recombination; some mutations inhibit the accumulation of Holliday structures, others inhibit their resolution to recombinant products. To see if heterology also alters the location of the strand exchange points, we determined the segregation pattern of three single and one multiple base pair substitution mutations of the overlap region in crosses with wild type sites. The mutations are known to differ in the severity of their recombination defect and in the stage of strand exchange they affect. The three single mutations behaved similarly: each segregated into both products of recombination, and the two products of a single crossover were frequently nonreciprocal in the overlap region. In contrast, the multiple mutation preferentially segregated into one of the two recombinant products, and the two products of a single crossover appeared to be fully reciprocal. The simplest explanation of the segregation pattern of the single mutations is that strand exchanges occur at the normal locations to produce recombinants with mismatched base pairs that are frequently repaired. The segregation pattern of the multiple mutation is consistent with the view that both strand exchanges usually occur to one side of the mutant site. We suggest that the segregation pattern of a particular mutation is determined by which stage of strand exchange it inhibits and by the severity of the inhibition.

Purification and characterization of the M.RsrI DNA methyltransferase from Escherichia coli.

The gene (rsrIM) encoding the RsrI DNA methyltransferase (M.RsrI) from Rhodobacter sphaeroides was cloned and expressed in Escherichia coli. Under the control of a bacteriophage T7 promoter, 2% of the total protein in a crude extract was M.RsrI. This level of expression represents an approximately 50-fold increase over that present in the natural host. Chromatography using DNA cellulose and the S-adenosylmethionine analogue, sinefungin, was useful in purifying the enzyme to homogeneity. The purification yielded 100 times more enzyme than was obtained from the same quantity of R. sphaeroides cell paste. M.RsrI deposits one methyl group per productive DNA-binding event, as does its functional but sequence-nonhomologous analogue, M.EcoRI. Unlike M.EcoRI, the R. sphaeroides enzyme is a dimer at micromolar concentrations.

Selection of mutations altering specificity in restriction-modification enzymes using the bacteriophage P22 challenge-phage system.

A method for selecting mutants of site-specific DNA-binding proteins has been applied to the study of the EcoRI and RsrI restriction-modification enzymes. Catalytically inactive variants of both endonucleases are shown to function as pseudo-repressors in the bacteriophage P22 challenge-phage assay, and, upon further mutagenesis of the gene encoding R.EcoRI, a variant of that enzyme has been selected which appears to bind EcoRI-methylated GAATTC sequences to the exclusion of unmethylated sites: this specificity is the opposite of that belonging to the native enzyme. Variants of the EcoRI methylase have also been found that lack either catalytic activity or both binding and catalytic activities.

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.

Mutants of Escherichia coli integration host factor: DNA-binding and recombination properties.

Integration host factor (IHF) is a protein encoded by Escherichia coli, which was first discovered as a requirement for bacteriophage lambda site-specific recombination. In this study, we characterized mutants of IHF for their ability to bind to various IHF binding sites in vivo and to promote recombination of lambda in vitro. DNA-binding in vivo was monitored using the challenge-phage system. If IHF binds to its DNA-binding site that has been placed into the P(ant) region of bacteriophage P22, it acts as a repressor of the ant (antirepressor) gene, leading to the formation of lysogens of Salmonella typhimurium. If IHF cannot bind to its site, antirepressor is made leading to cell lysis. Challenge phages containing chimeras of different lambda IHF binding sites were constructed to test the contribution to the binding of a dA+dT-rich region, found in the sequence of the H' site but not in the H' site. In one case, the binding of mutant IHF proteins was enhanced by the presence of the dA+dT-rich region, indicating that IHF may be affected by neighboring bases and local DNA structure when it binds to its site. A subset of the mutant proteins retained the ability to form a looped attL complex in vivo, representing part of a higher-order protein-DNA complex (the 'intasome'). Additionally, this same subset of proteins also promoted the integration and excision of bacteriophage lambda in vitro. Thus, these mutant proteins not only retain their DNA-bending ability but make any protein-protein contacts necessary to form a recombination-proficient intasome.

Restriction endonuclease RsrI from Rhodobacter sphaeroides, an isoschizomer of EcoRI: purification and properties.

We have purified RsrI endonuclease (R.RsrI), an isoschizomer of EcoRI, from Rhodobacter sphaeroides strain 630. The enzyme is homogeneous as judged by polyacrylamide gel electrophoresis and size-exclusion high-performance liquid chromatography. RsrI endonuclease is a dimer over the concentration range of 0.05 to 1.4 mg/ml. The reduced and denatured molecular weight of the enzyme is 30,000 Da. R.RsrI, like R.EcoRI, catalyzes the cleavage of duplex DNA and oligodeoxyribonucleotides between the first two residues of the sequence GAATTC. R.RsrI exhibits a KM of 14 nM and a kcat of 6.5 min-1 when reacting with pBR322 DNA at 25 degrees C. R.RsrI differs from R.EcoRI in its N-terminal amino acid sequence, susceptibility to inhibition by antibodies, sensitivity to N-ethylmaleimide, isoelectric point, state of aggregation at high concentrations, temperature lability, and conditions for optimal reaction. R.RsrI displays a reduction of specificity ("star activity") under conditions that also relax the specificity of R.EcoRI.