Purification and characterization of bacteriophage P22 Xis protein.

The temperate bacteriophages lambda 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.

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.

The molecular basis of co-operative DNA binding between lambda integrase and excisionase.

Higher-order nucleoprotein complexes often stabilize catalytic proteins in appropriate conformations for optimal activity and contribute to regulation during reactions requiring association of proteins and DNA. Formation of such complexes, known as intasomes, is required for site-specific recombination catalysed by bacteriophage Lambda Integrase protein (Int). Int-catalysed recombination is regulated by a second bacteriophage-encoded protein, Excisionase (Xis), which both stimulates excision and inhibits integration.

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.

A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes.

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.

Conservation of structure and function among tyrosine recombinases: homology-based modeling of the lambda integrase core-binding domain.

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.

Regulation of site-specific recombination by the C-terminus of lambda integrase.

Site-specific recombination catalyzed by bacteriophage lambda 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.

Interactions between integrase and excisionase in the phage lambda excisive nucleoprotein complex.

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.

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.

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.

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.

Characterization of bacteriophage lambda excisionase mutants defective in DNA binding.

The bacteriophage lambda 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.

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.

Specificity determinants for bacteriophage Hong Kong 022 integrase: analysis of mutants with relaxed core-binding specificities.

The integrase (Int) proteins encoded by bacteriophages HK022 and lambda catalyse similar site-specific integration and excision reactions between specific DNA regions known as attachment (att) sites. However, the Int proteins of HK022 and lambda are unable to catalyse recombination between non-cognate att sites. The att sites of both phages contain weak binding sites for Int, known as 'core-type' sites.

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.

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.

Genetic analysis of second-site revertants of bacteriophage lambda integrase mutants.

Bacteriophage lambda site-specific recombination is catalyzed by the phage-encoded integrase (Int) protein. Using a collection of 21 recombination-defective Int mutants, we performed a second-site reversion analysis. One of the primary mutants contained a valine-to-glutamic acid change at position 175 (V175E), and a pseudorevertant with a lysine change at this site (V175K) was also isolated.

Mutational analysis of protein binding sites involved in formation of the bacteriophage lambda attL complex.

Bacteriophage lambda site-specific recombination requires the formation of higher-order protein-DNA complexes to accomplish synapsis of the partner attachment (att) sites as well as for the regulation of the integration and excision reactions. The att sites are composed of a core region, the actual site of strand exchange, and flanking arm regions. The attL site consists of two core sites (C and C'), an integration host factor (IHF) binding site (H'), and three contiguous Int binding arm sites (P'1, P'2, and P'3).

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.

Examining the contribution of a dA+dT element to the conformation of Escherichia coli integration host factor-DNA complexes.

DNA binding proteins that induce structural changes in DNA are common in both prokaryotes and eukaryotes. Integration host factor (IHF) is a multi-functional DNA binding and bending protein of Escherichia coli that can mediate protein-protein and protein-DNA interactions by bending DNA. Previously we have shown that the presence of a dA+dT element 5'-proximal to an IHF consensus sequence can affect the binding of IHF to a particular site.

Transcription termination at the Escherichia coli thra terminator by spinach chloroplast RNA polymerase in vitro is influenced by downstream DNA sequences.

We have investigated the mechanism of transcription termination in vitro by spinach chloroplast RNA polymerase using templates encoding variants of the transcription-termination structure (attenuator) of the regulatory region of the threonine (thr) operon of Escherichia coli. Fourteen sequence variants located within its d(G+C) stem-loop and d(A+T)-rich regions were studied. We found that the helix integrity in the stem-loop structure is necessary for termination but that its stability is not directly correlated with termination efficiency.

The specific binding of Escherichia coli integration host factor involves both major and minor grooves of DNA.

The integration host factor (IHF) of Escherichia coli is a small, sequence-specific DNA-binding protein. The specific and nonspecific binding constants of IHF were estimated by gel-retardation assays. The equilibrium association constant of IHF for the H' site in lambda attP is 6.

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.

Determining the DNA sequence elements required for binding integration host factor to two different target sites.

Binding sites for the Escherichia coli protein integration host factor (IHF) include a set of conserved bases that can be summarized by the consensus sequence WATCAANNNNTTR (W is dA or dT, R is dA or dG, and N is any nucleotide). However, additional 5'-proximal bases, whose common feature is a high dA+dT content, are also thought to be required for binding at some sites. We examine the relative contribution of these two sequence elements to IHF binding to the H' and H1 sites in attP of bacteriophage lambda by using the bacteriophage P22-based challenge-phage system.

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.