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1.  Purification and Characterization of Bacteriophage P22 Xis Protein▿  
Journal of Bacteriology  2008;190(17):5781-5796.
The temperate bacteriophages λ and P22 share similarities in their site-specific recombination reactions. Both require phage-encoded integrase (Int) proteins for integrative recombination and excisionase (Xis) proteins for excision. These proteins bind to core-type, arm-type, and Xis binding sites to facilitate the reaction. λ and P22 Xis proteins are both small proteins (λ Xis, 72 amino acids; P22 Xis, 116 amino acids) and have basic isoelectric points (for P22 Xis, 9.42; for λ Xis, 11.16). However, the P22 Xis and λ Xis primary sequences lack significant similarity at the amino acid level, and the linear organizations of the P22 phage attachment site DNA-binding sites have differences that could be important in quaternary intasome structure. We purified P22 Xis and studied the protein in vitro by means of electrophoretic mobility shift assays and footprinting, cross-linking, gel filtration stoichiometry, and DNA bending assays. We identified one protected site that is bent approximately 137 degrees when bound by P22 Xis. The protein binds cooperatively and at high protein concentrations protects secondary sites that may be important for function. Finally, we aligned the attP arms containing the major Xis binding sites from bacteriophages λ, P22, L5, HP1, and P2 and the conjugative transposon Tn916. The similarity in alignments among the sites suggests that Xis-containing bacteriophage arms may form similar structures.
doi:10.1128/JB.00170-08
PMCID: PMC2519534  PMID: 18502866
2.  Dimerization of the bacterial RsrI N6-adenine DNA methyltransferase 
Nucleic Acids Research  2006;34(3):806-815.
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 Å2 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 β-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.
doi:10.1093/nar/gkj486
PMCID: PMC1361615  PMID: 16464821
3.  SURVEY AND SUMMARY: A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes 
Nucleic Acids Research  2003;31(7):1805-1812.
A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.
PMCID: PMC152790  PMID: 12654995
4.  SURVEY AND SUMMARY: Conservation of structure and function among tyrosine recombinases: homology-based modeling of the lambda integrase core-binding domain 
Nucleic Acids Research  2003;31(3):805-818.
Tyrosine recombinases participate in diverse biological processes by catalyzing recombination between specific DNA sites. Although a conserved protein fold has been described for the catalytic (CAT) domains of five recombinases, structural relationships between their core-binding (CB) domains remain unclear. Despite differences in the specificity and affinity of core-type DNA recognition, a conserved binding mechanism is suggested by the shared two-domain motif in crystal structure models of the recombinases Cre, XerD and Flp. We have found additional evidence for conservation of the CB domain fold. Comparison of XerD and Cre crystal structures showed that their CB domains are closely related; the three central α-helices of these domains are superposable to within 1.44 Å. A structure-based multiple sequence alignment containing 25 diverse CB domain sequences provided evidence for widespread conservation of both structural and functional elements in this fold. Based upon the Cre and XerD crystal structures, we employed homology modeling to construct a three-dimensional structure for the λ integrase CB domain. The model provides a conceptual framework within which many previously identified, functionally important amino acid residues were investigated. In addition, the model predicts new residues that may participate in core-type DNA binding or dimerization, thereby providing hypotheses for future genetic and biochemical experiments.
PMCID: PMC149183  PMID: 12560475
5.  Regulation of site-specific recombination by the C-terminus of λ integrase 
Nucleic Acids Research  2002;30(23):5193-5204.
Site-specific recombination catalyzed by bacteriophage λ integrase (Int) is essential for establishment and termination of the viral lysogenic life cycle. Int is the archetype of the tyrosine recombinase family whose members are responsible for DNA rearrangement in prokaryotes, eukaryotes and viruses. The mechanism regulating catalytic activity during recombination is incompletely understood. Studies of tyrosine recombinases bound to their target substrates suggest that the C-termini of the proteins are involved in protein–protein contacts that control the timing of DNA cleavage events during recombination. We investigated an Int truncation mutant (W350) that possesses enhanced topoisomerase activity but greater than 100-fold reduced recombination activity. Alanine scanning mutagenesis of the C-terminus indicates that two mutants, W350A and I353A, cannot perform site-specific recombination although their DNA binding, cleavage and ligation activities are at wild-type levels. Two other mutants, R346A and R348A, are deficient solely in the ability to cleave DNA. To explain these results, we have constructed a homology-threaded model of the Int structure using a Cre crystal structure. We propose that residues R346 and R348 are involved in orientation of the catalytic tyrosine that cleaves DNA, whereas W350 and I353 control and make intermolecular contacts with other Int proteins in the higher order recombination structures known as intasomes. These results suggest that Int and the other tyrosine recombinases have evolved regulatory contacts that coordinate site-specific recombination at the C-terminus.
PMCID: PMC137966  PMID: 12466544
6.  Interactions between Integrase and Excisionase in the Phage Lambda Excisive Nucleoprotein Complex 
Journal of Bacteriology  2002;184(18):5200-5203.
Bacteriophage lambda site-specific recombination comprises two overall reactions, integration into and excision from the host chromosome. Lambda integrase (Int) carries out both reactions. During excision, excisionase (Xis) helps Int to bind DNA and introduces a bend in the DNA that facilitates formation of the proper excisive nucleoprotein complex. The carboxyl-terminal α-helix of Xis is thought to interact with Int through direct protein-protein interactions. In this study, we used gel mobility shift assays to show that the amino-terminal domain of Int maintained cooperative interactions with Xis. This finding indicates that the amino-terminal arm-type DNA binding domain of Int interacts with Xis.
doi:10.1128/JB.184.18.5200-5203.2002
PMCID: PMC135313  PMID: 12193639
7.  DNA binding properties in vivo and target recognition domain sequence alignment analyses of wild-type and mutant RsrI [N6-adenine] DNA methyltransferases 
Nucleic Acids Research  2000;28(20):3972-3981.
A genetic selection method, the P22 challenge-phage assay, was used to characterize DNA binding in vivo by the prokaryotic β class [N6-adenine] DNA methyltransferase M·RsrI. M·RsrI mutants with altered binding affinities in vivo were isolated. Unlike the wild-type enzyme, a catalytically compromised mutant, M·RsrI (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·RsrI (L72P/D173A), showed less binding in vivo than did M·RsrI (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 β 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 (α and γ) having the same DNA recognition sequence as the β class MTases share related regions of amino acid sequences in their TRDs.
PMCID: PMC110778  PMID: 11024177
8.  Substrate binding in vitro and kinetics of RsrI [N6-adenine] DNA methyltransferase 
Nucleic Acids Research  2000;28(20):3962-3971.
RsrI [N6-adenine] DNA methyltransferase (M·RsrI), 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·RsrI 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·RsrI binding was affected by the methylation status of the DNA substrate and was enhanced by the presence of the cofactor analog. M·RsrI 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·RsrI extruding the target base from the duplex. Consistent with such base flipping, an ∼1.7-fold fluorescence intensity increase was observed upon stoichiometric addition of M·RsrI 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·RsrI–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.
PMCID: PMC110777  PMID: 11024176
9.  Structure of RsrI methyltransferase, a member of the N6-adenine β class of DNA methyltransferases 
Nucleic Acids Research  2000;28(20):3950-3961.
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 N6-adenine methyltransferase belonging to the β class of bacterial methyltransferases is described here. The structure of M·RsrI from Rhodobacter sphaeroides, which methylates the second adenine of the GAATTC sequence, was determined to 1.75 Å 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 PvuII methyltransferase, a cytosine amino methyltransferase of the same β group. The larger nucleotide binding pocket observed in M·RsrI is expected because it methylates adenine. However, the most striking difference between the RsrI 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.
PMCID: PMC110776  PMID: 11024175
10.  Characterization of Bacteriophage Lambda Excisionase Mutants Defective in DNA Binding 
Journal of Bacteriology  2000;182(20):5807-5812.
The bacteriophage λ excisionase (Xis) is a sequence-specific DNA binding protein required for excisive recombination. Xis binds cooperatively to two DNA sites arranged as direct repeats on the phage DNA. Efficient excision is achieved through a cooperative interaction between Xis and the host-encoded factor for inversion stimulation as well as a cooperative interaction between Xis and integrase. The secondary structure of the Xis protein was predicted to contain a typical amphipathic helix that spans residues 18 to 28. Several mutants, defective in promoting excision in vivo, were isolated with mutations at positions encoding polar amino acids in the putative helix (T. E. Numrych, R. I. Gumport, and J. F. Gardner, EMBO J. 11:3797–3806, 1992). We substituted alanines for the polar amino acids in this region. Mutant proteins with substitutions for polar amino acids in the amino-terminal region of the putative helix exhibited decreased excision in vivo and were defective in DNA binding. In addition, an alanine substitution at glutamic acid 40 also resulted in altered DNA binding. This indicates that the hydrophilic face of the α-helix and the region containing glutamic acid 40 may form the DNA binding surfaces of the Xis protein.
PMCID: PMC94704  PMID: 11004181

Results 1-10 (10)