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Architectural DNA-binding proteins (ADBPs) are abundant constituents of eukaryotic or bacterial chromosomes that bind DNA promiscuously and function in diverse DNA reactions. They generate large conformational changes in DNA upon binding yet can slide along DNA when searching for functional binding sites. Here we investigate the mechanism by which ADBPs diffuse on DNA by single-molecule analyses of mutant proteins rationally chosen to distinguish between rotation-coupled diffusion and DNA surface sliding after transient unbinding from the groove(s). The properties of yeast Nhp6A mutant proteins, combined with molecular dynamics simulations, suggest Nhp6A switches between two binding modes: a static state, in which the HMGB domain is bound within the minor groove with the DNA highly bent, and a mobile state, where the protein is traveling along the DNA surface by means of its flexible N-terminal basic arm. The behaviors of Fis mutants, a bacterial nucleoid-associated helix-turn-helix dimer, are best explained by mobile proteins unbinding from the major groove and diffusing along the DNA surface. Nhp6A, Fis, and bacterial HU are all near exclusively associated with the chromosome, as packaged within the bacterial nucleoid, and can be modeled by three diffusion modes where HU exhibits the fastest and Fis the slowest diffusion.

DNA binding proteins rapidly locate their specific DNA targets through a combination of 3D and 1D diffusion mechanisms, with the 1D search involving bidirectional sliding along DNA. However, even in nucleosome-free regions, chromosomes are highly decorated with associated proteins that may block sliding. Here we investigate the ability of the abundant chromatin-associated HMGB protein Nhp6A from Saccharomyces cerevisiae to travel along DNA in the presence of other architectural DNA binding proteins using single-molecule fluorescence microscopy. We observed that 1D diffusion by Nhp6A molecules is retarded by increasing densities of the bacterial proteins Fis and HU and by Nhp6A, indicating these structurally diverse proteins impede Nhp6A mobility on DNA. However, the average travel distances were larger than the average distances between neighboring proteins, implying Nhp6A is able to bypass each of these obstacles. Together with molecular dynamics simulations, our analyses suggest two binding modes: mobile molecules that can bypass barriers as they seek out DNA targets, and near stationary molecules that are associated with neighboring proteins or preferred DNA structures. The ability of mobile Nhp6A molecules to bypass different obstacles on DNA suggests they do not block 1D searches by other DNA binding proteins.

Bacteriophage serine integrases catalyze highly specific recombination reactions between defined DNA segments called att sites. These reactions are reversible depending upon the presence of a second phage-encoded directionality factor. The bipartite C-terminal DNA binding region of integrases includes a recombinase domain (RD) connected to a zinc-binding domain (ZD), which contains a long flexible coiled-coil (CC) motif that extends away from the bound DNA. We directly show that the identities of the phage A118 integrase att sites are specified by the DNA spacing between the RD and ZD DNA recognition determinants, which in turn, directs the relative trajectories of the CC motifs on each subunit of the att -bound integrase dimer. Recombination between compatible dimer-bound att sites requires minimal length CC motifs and 14 residues surrounding the tip where pairing of CC motifs between synapsing dimers occurs. Our alanine-scanning data suggests that molecular interactions between CC motif tips may differ in integrative ( attP x attB ) and excisive ( attL x attR ) recombination reactions. We identify mutations in 5 residues within the integrase oligomerization helix that control the remodeling of dimers into tetramers during synaptic complex formation. Whereas most of these gain-of-function mutants still require the CC motifs for synapsis, one mutant efficiently, but indiscriminantly, forms synaptic complexes without the CC motifs. However, the CC motifs are still required for recombination, suggesting a function for the CC motifs after initial assembly of the integrase synaptic tetramer. Importance The robust and exquisitely-regulated site-specific recombination reactions promoted by serine integrases are integral to the life cycle of temperate bacteriophage, and in the case of the A118 prophage, are an important virulence factor by Listeria monocytogenes . The properties of these recombinases have led to their repurposing into tools for genetic engineering and synthetic biology. In this report, we identify determinants regulating synaptic complex formation between correct DNA sites, including the DNA architecture responsible for specifying the identity of recombination sites, features of the unique coiled-coil structure on the integrase that are required to initiate synapsis, and amino acid residues on the integrase oligomerization helix that control the remodeling of synapsing dimers into a tetramer active for DNA strand exchange.

Localized arrays of proteins cooperatively assemble onto chromosomes to control DNA activity in many contexts. Binding cooperativity is often mediated by specific protein–protein interactions, but cooperativity through DNA structure is becoming increasingly recognized as an additional mechanism. During the site-specific DNA recombination reaction that excises phage λ from the chromosome, the bacterial DNA architectural protein Fis recruits multiple λ-encoded Xis proteins to the attR recombination site. Here, we report X-ray crystal structures of DNA complexes containing Fis + Xis, which show little, if any, contacts between the two proteins. Comparisons with structures of DNA complexes containing only Fis or Xis, together with mutant protein and DNA binding studies, support a mechanism for cooperative protein binding solely by DNA allostery. Fis binding both molds the minor groove to potentiate insertion of the Xis β-hairpin wing motif and bends the DNA to facilitate Xis-DNA contacts within the major groove. The Fis-structured minor groove shape that is optimized for Xis binding requires a precisely positioned pyrimidine-purine base-pair step, whose location has been shown to modulate minor groove widths in Fis-bound complexes to different DNA targets.

Architectural DNA-binding proteins function to regulate diverse DNA reactions and have the defining property of significantly changing DNA conformation. Although the 1D movement along DNA by other types of DNA-binding proteins has been visualized, the mobility of architectural DNA-binding proteins on DNA remains unknown. Here, we applied single-molecule fluorescence imaging on arrays of extended DNA molecules to probe the binding dynamics of three structurally distinct architectural DNA-binding proteins: Nhp6A, HU, and Fis. Each of these proteins was observed to move along DNA, and the salt concentration independence of the 1D diffusion implies sliding with continuous contact to DNA. Nhp6A and HU exhibit a single sliding mode, whereas Fis exhibits two sliding modes. Based on comparison of the diffusion coefficients and sizes of many DNA binding proteins, the architectural proteins are categorized into a new group distinguished by an unusually high free-energy barrier for 1D diffusion. The higher free-energy barrier for 1D diffusion by architectural proteins can be attributed to the large DNA conformational changes that accompany binding and impede rotation-coupled movement along the DNA grooves.

The serine integrase of phage A118 catalyzes integrative recombination between attP on the phage and a specific attB locus on the chromosome of Listeria monocytogenes , but it is unable to promote excisive recombination between the hybrid attL and attR sites found on the integrated prophage without assistance by a recombination directionality factor (RDF). We have identified and characterized the phage-encoded RDF Gp44, which activates the A118 integrase for excision and inhibits integration. Gp44 binds to the C-terminal DNA binding domain of integrase, and we have localized the primary binding site to be within the mobile coiled-coil (CC) motif but distinct from the distal tip of the CC that is required for recombination. This interaction is sufficient to inhibit integration, but a second interaction involving the N-terminal end of Gp44 is also required to activate excision. We provide evidence that these two contacts modulate the trajectory of the CC motifs as they extend out from the integrase core in a manner dependent upon the identities of the four att sites. Our results support a model whereby Gp44 shapes the Int-bound complexes to control which att sites can synapse and recombine. IMPORTANCE Serine integrases mediate directional recombination between bacteriophage and bacterial chromosomes. These highly regulated site-specific recombination reactions are integral to the life cycle of temperate phage and, in the case of Listeria monocytogenes lysogenized by A118 family phage, are an essential virulence determinant. Serine integrases are also utilized as tools for genetic engineering and synthetic biology because of their exquisite unidirectional control of the DNA exchange reaction. Here, we identify and characterize the recombination directionality factor (RDF) that activates excision and inhibits integration reactions by the phage A118 integrase. We provide evidence that the A118 RDF binds to and modulates the trajectory of the long coiled-coil motif that extends from the large carboxyl-terminal DNA binding domain and is postulated to control the early steps of recombination site synapsis.

The binding of transcription factors (TFs) to DNA controls most aspects of cellular function, making the understanding of their binding kinetics imperative. The standard description of bimolecular interactions posits TF off-rates are independent of TF concentration in solution. However, recent observations have revealed that proteins in solution can accelerate the dissociation of DNA-bound proteins. To study the molecular basis of facilitated dissociation (FD), we have used single-molecule imaging to measure dissociation kinetics of Fis, a key E. coli TF and major bacterial nucleoid protein, from single dsDNA binding sites. We observe a strong FD effect characterized by an exchange rate ∽1 × 104 M-1s-1, establishing that FD of Fis occurs at the single-binding-site level, and we find that the off-rate saturates at large Fis concentrations in solution. While spontaneous (i.e., competitor-free) dissociation shows a strong salt dependence, we find that facilitated dissociation depends only weakly on salt. These results are quantitatively explained by a model in which partially dissociated bound proteins are susceptible to invasion by competitor proteins in solution. We also report FD of NHP6A, a yeast TF whose structure differs significantly from Fis. We further perform molecular dynamics simulations, which indicate that FD can occur for molecules that interact far more weakly than those we have studied. Taken together, our results indicate that FD is a general mechanism assisting in the local removal of TFs from their binding sites and does not necessarily require cooperativity, clustering, or binding site overlap.SIGNIFICANCE STATEMENTTranscription factors (TFs) control biological processes by binding and unbinding to DNA. Therefore it is crucial to understand the mechanisms that affect TF binding kinetics. Recent studies challenge the standard picture of TF binding kinetics by demonstrating cases of proteins in solution accelerating TF dissociation rates through a facilitated dissociation (FD) process. Our study shows that FD can occur at the level of single binding sites, without the action of large protein clusters or long DNA segments. Our results quantitatively support a model of FD in which competitor proteins invade partially dissociated states of DNA-bound TFs. FD is expected to be a general mechanism for modulating gene expression by altering the occupancy of TFs on the genome.Author ContributionsRamsey I. Kamardesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperEdward J. Banigandesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperAykut Erbasdesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperRebecca D. Giuntolidesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperMonica Olvera de la Cruzdesigned research, performed research, wrote the paperReid C. Johnsondesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperJohn F. Markodesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paper

Off-rates of proteins from the DNA double helix are widely considered to be dependent only on the interactions inside the initially bound protein-DNA complex and not on the concentration of nearby molecules. However, a number of recent single-DNA experiments have shown off-rates that depend on solution protein concentration, or “facilitated dissociation.” Here, we demonstrate that this effect occurs for the major Escherichia coli nucleoid protein Fis on isolated bacterial chromosomes. We isolated E. coli nucleoids and showed that dissociation of green fluorescent protein (GFP)-Fis is controlled by solution Fis concentration and exhibits an “exchange” rate constant ( k exch ) of ≈10 4 M −1 s −1 , comparable to the rate observed in single-DNA experiments. We also show that this effect is strongly salt dependent. Our results establish that facilitated dissociation can be observed in vitro on chromosomes assembled in vivo . IMPORTANCE Bacteria are important model systems for the study of gene regulation and chromosome dynamics, both of which fundamentally depend on the kinetics of binding and unbinding of proteins to DNA. In experiments on isolated E. coli chromosomes, this study showed that the prolific transcription factor and chromosome packaging protein Fis displays a strong dependence of its off-rate from the bacterial chromosome on Fis concentration, similar to that observed in in vitro experiments. Therefore, the free cellular DNA-binding protein concentration can strongly affect lifetimes of proteins bound to the chromosome and must be taken into account in quantitative considerations of gene regulation. These results have particularly profound implications for transcription factors where DNA binding lifetimes can be a critical determinant of regulatory function.

DNA strand exchange by serine recombinases has been proposed to occur by a large-scale rotation of halves of the recombinase tetramer. Here we provide the first direct physical evidence for the subunit rotation mechanism for the Hin serine invertase. Single-DNA looping assays using an activated mutant (Hin-H107Y) reveal specific synapses between two hix sites. Two-DNA “braiding” experiments, where separate DNA molecules carrying a single hix are interwound, show that Hin-H107Y cleaves both hix sites and mediates multi-step rotational relaxation of the interwinding. The variable numbers of rotations in the DNA braid experiments are in accord with data from bulk experiments that follow DNA topological changes accompanying recombination by the hyperactive enzyme. The relatively slow Hin rotation rates, combined with pauses, indicate considerable rotary friction between synapsed subunit pairs. A rotational pausing mechanism intrinsic to serine recombinases is likely to be crucial for DNA ligation and for preventing deleterious DNA rearrangements.

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequences in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. The affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.

Summary We examine whether the Escherichia coli chromosome is folded into a self-adherent nucleoprotein complex, or alternately is a confined but otherwise unconstrained self-avoiding polymer. We address this through in vivo visualization, using an inducible GFP fusion to the nucleoid-associated protein Fis to nonspecifically decorate the entire chromosome. For a range of different growth conditions, the chromosome is a compact structure that does not fill the volume of the cell, and which moves from the new pole to the cell centre.Duringrapidgrowth,chromosomesegregation occurs well before cell division, with daughter chromosomes coupled by a thin inter-daughter filament before complete segregation, whereas during slow growth chromosomes stay adjacent until cell division occurs. Image correlation analysis indicates that sub-nucleoid structure is stable on a 1 min timescale, comparable to the timescale for redistribution time measured for GFP‐Fis after photobleaching. Optical deconvolutionandwrithecalculationanalysisindicate that the nucleoid has a large-scale coiled organization rather than being an amorphous mass. Our observations are consistent with the chromosome having a self-adherent filament organization.

Chromosomes contain DNA covered with proteins performing functions such as architectural organization and transcriptional regulation. The ability to count the number of proteins bound to various regions of the genome is essential for understanding both architectural and regulatory functions. We present a straightforward method of counting gfp-conjugated proteins bound to an individual duplex DNA molecule by calibrating to a commercially available fluorescence standard using wide-field fluorescence microscopy. We demonstrate our method using the E. coli nucleoid-associated protein Fis.

Serine recombinases, which generate double-strand breaks in DNA, must be carefully regulated to ensure that chemically active DNA complexes are assembled correctly. In the Hin-catalyzed site-specific DNA inversion reaction, two inversely oriented recombination sites on the same DNA molecule assemble into a synaptic complex that uniquely generates inversion products. The Fis-bound recombinational enhancer, together with topological constraints directed by DNA supercoiling, functions to regulate Hin synaptic complex formation and activity. We have isolated a collection of gain-of-function mutants in 22 positions within the catalytic and oligomerization domains of Hin using two genetic screens and by site-directed mutagenesis. One genetic screen measured recombination in the absence of Fis and the other assessed SOS induction as a readout of increased DNA cleavage. These mutations, together with molecular modeling, identify important sites of dynamic intrasubunit and intersubunit interactions that regulate assembly of the active tetrameric recombination complex. Of particular interest are interactions between the oligomerization helix (helix E) and the catalytic domain of the same subunit that function to hold the dimer in an inactive state in the absence of the Fis/enhancer system. Among these is a relay involving a triad of phenylalanines that are proposed to switch positions during the transition from dimers to the catalytically active tetramer. Novel Hin mutants that generate synaptic complexes that are blocked at steps prior to DNA cleavage are also described.

The multistep kinetics through which DNA-binding proteins bind their targets are heavily studied, but relatively little attention has been paid to proteins leaving the double helix. Using single-DNA stretching and fluorescence detection, we find that sequence-neutral DNA-binding proteins Fis, HU and NHP6A readily exchange with themselves and with each other. In experiments focused on the Escherichia coli nucleoid-associated protein Fis, only a small fraction of protein bound to DNA spontaneously dissociates into protein-free solution. However, if Fis is present in solution, we find that a concentration-dependent exchange reaction occurs which turns over the bound protein, with a rate of k(exch) = 6 × 10(4) M(-1)s(-1). The bacterial DNA-binding protein HU and the yeast HMGB protein NHP6A display the same phenomenon of protein in solution accelerating dissociation of previously bound labeled proteins as exchange occurs. Thus, solvated proteins can play a key role in facilitating removal and renewal of proteins bound to the double helix, an effect that likely plays a major role in promoting the turnover of proteins bound to DNA in vivo and, therefore, in controlling the dynamics of gene regulation.

The Saccharomyces cerevisiae protein Nhp6A is a model for the abundant and multifunctional high-mobility group B (HMGB) family of chromatin-associated proteins. Nhp6A binds DNA in vitro without sequence specificity and bends DNA sharply, but its role in chromosome biology is poorly understood. We show by whole-genome chromatin immunoprecipitation (ChIP) and high-resolution whole-genome tiling arrays (ChIP–chip) that Nhp6A is localized to specific regions of chromosomes that include ∼23% of RNA polymerase II promoters. Nhp6A binding functions to stabilize nucleosomes, particularly at the transcription start site of these genes. Both genomic binding and transcript expression studies point to functionally related groups of genes that are bound specifically by Nhp6A and whose transcription is altered by the absence of Nhp6. Genomic analyses of Nhp6A mutants specifically defective in DNA bending reveal a critical role of DNA bending for stabilizing chromatin and coregulation of transcription but not for targeted binding by Nhp6A. We conclude that the chromatin environment, not DNA sequence recognition, localizes Nhp6A binding, and that Nhp6A stabilizes chromatin structure and coregulates transcription.

HU is one of the most abundant proteins in bacterial chromosomes and participates in nucleoid compaction and gene regulation. We report experiments using DNA stretching that study the dependence of DNA condensation by HU on force, salt and HU concentration. Previous experiments at sub-physiological salt levels revealed that low concentrations of HU could compact DNA, whereas larger HU concentrations formed a DNA-stiffening complex. Here we report that this bimodal binding behavior depends sensitively on salt concentration. Only the compaction mode was observed for 150 mM and higher NaCl levels, i.e. for physiological salt concentrations. Similar results were obtained for the more physiological salt K-glutamate. Real-time studies of dissociation kinetics revealed that HU unbound slowly (minutes to hours under the conditions studied) but completely for salt concentrations at or above 100 mM NaCl; the lifetime of HU complexes was observed to increase with the HU concentration at which the complexes were formed, and to decrease with salt concentration. Higher salt levels of 300 mM NaCl completely eliminated observable HU binding to DNA. Finally, we observed that the dissociation kinetics depend on force applied to the DNA: increased applied force in the sub-piconewton range accelerates dissociation, suggesting a mechanism for DNA tension to regulate chromosome structure and gene expression.

The bacterial nucleoid-associated protein Fis regulates diverse reactions by bending DNA and through DNA-dependent interactions with other control proteins and enzymes. In addition to dynamic nonspecific binding to DNA, Fis forms stable complexes with DNA segments that share little sequence conservation. Here we report the first crystal structures of Fis bound to high- and low-affinity 27-base-pair DNA sites. These 11 structures reveal that Fis selects targets primarily through indirect recognition mechanisms involving the shape of the minor groove and sequence-dependent induced fits over adjacent major groove interfaces. The DNA shows an overall curvature of approximately 65 degrees , and the unprecedented close spacing between helix-turn-helix motifs present in the apodimer is accommodated by severe compression of the central minor groove. In silico DNA structure models show that only the roll, twist, and slide parameters are sufficient to reproduce the changes in minor groove widths and recreate the curved Fis-bound DNA structure. Models based on naked DNA structures suggest that Fis initially selects DNA targets with intrinsically narrow minor grooves using the separation between helix-turn-helix motifs in the Fis dimer as a ruler. Then Fis further compresses the minor groove and bends the DNA to generate the bound structure.

Hin, a member of the serine family of site-specific recombinases, regulates gene expression by inverting a DNA segment. DNA inversion requires assembly of an invertasome complex in which a recombinational enhancer DNA segment bound by the Fis protein associates with the Hin synaptic complex at the base of a supercoiled DNA branch. Each of the four Hin subunits becomes covalently joined to the cleaved DNA ends, and DNA exchange occurs by translocation of a Hin subunit pair within the tetramer. We show here that, although the Hin tetramer forms a bidirectional molecular swivel, the Fis/enhancer system determines both the direction and number of subunit rotations. The chirality of supercoiling directs rotational direction, and the short DNA loop stabilized by Fis-Hin contacts limit rotational processivity, thereby ensuring that the DNA strands religate in the recombinant configuration. We identify multiple rotational conformers that are formed under different supercoiling and solution conditions.

The phage-encoded Xis protein is the major determinant controlling the direction of recombination in phage lambda. Xis is a winged-helix DNA binding protein that cooperatively binds to the attR recombination site to generate a curved microfilament, which promotes assembly of the excisive intasome but inhibits formation of an integrative intasome. We find that lambda synthesizes surprisingly high levels of Xis immediately upon prophage induction when excision rates are maximal. However, because of its low sequence-specific binding activity, exemplified by a 1.9 Å co-crystal structure of a nonspecifically bound DNA complex, Xis is relatively ineffective at promoting excision in vivo in the absence of the host Fis protein. Fis binds to a segment in attR that almost entirely overlaps one of the Xis binding sites. Instead of sterically excluding Xis binding from this site, as has been previously believed, we show that Fis enhances binding of all three Xis protomers to generate the microfilament. A specific Fis-Xis interface is supported by the effects of mutations within each protein, and relaxed, but not completely sequence-neutral, binding by the central Xis protomer is supported by the effects of DNA mutations. We present a structural model for the 50 bp curved Fis-Xis cooperative complex that is assembled between the arm and Holliday junction Int binding sites whose trajectory places constraints on models for the excisive intasome structure.

The DNA architectural protein Xis regulates the construction of higher-order nucleoprotein intasomes that integrate and excise the genome of phage lambda from the Escherichia coli chromosome. Xis modulates the directionality of site-specific recombination by stimulating phage excision 10 6 -fold, while simultaneously inhibiting phage reintegration. Control is exerted by cooperatively assembling onto a ≈35-bp DNA regulatory element, which it distorts to preferentially stabilize an excisive intasome. Here, we report the 2.6-Å crystal structure of the complex between three cooperatively bound Xis proteins and a 33-bp DNA containing the regulatory element. Xis binds DNA in a head-to-tail orientation to generate a micronucleoprotein filament. Although each protomer is anchored to the duplex by a similar set of nonbase specific contacts, malleable protein–DNA interactions enable binding to sites that differ in nucleotide sequence. Proteins at the ends of the duplex sequence specifically recognize similar binding sites and participate in cooperative binding via protein–protein interactions with a bridging Xis protomer that is bound in a less specific manner. Formation of this polymer introduces ≈72° of curvature into the DNA with slight positive writhe, which functions to connect disparate segments of DNA bridged by integrase within the excisive intasome.

The rate of dissociation of a DNA-protein complex is often considered to be a property of that complex, without dependence on other nearby molecules in solution. We study the kinetics of dissociation of the abundant Escherichia coli nucleoid protein Fis from DNA, using a single-molecule mechanics assay. The rate of Fis dissociation from DNA is strongly dependent on the solution concentration of DNA. The off-rate (k(off)) of Fis from DNA shows an initially linear dependence on solution DNA concentration, characterized by an exchange rate of k(ex)≈9×10(-4) (ng/μl)(-1) s(-1) for 100 mM univalent salt buffer, with a very small off-rate at zero DNA concentration. The off-rate saturates at approximately k(off,max)≈8×10(-3) s(-1) for DNA concentrations above ≈20 ng/μl. This exchange reaction depends mainly on DNA concentration with little dependence on the length of the DNA molecules in solution or on binding affinity, but this does increase with increasing salt concentration. We also show data for the yeast HMGB protein NHP6A showing a similar DNA-concentration-dependent dissociation effect, with faster rates suggesting generally weaker DNA binding by NHP6A relative to Fis. Our results are well described by a model with an intermediate partially dissociated state where the protein is susceptible to being captured by a second DNA segment, in the manner of "direct transfer" reactions studied for other DNA-binding proteins. This type of dissociation pathway may be important to protein-DNA binding kinetics in vivo where DNA concentrations are large.

Two critical steps controlling serine recombinase activity are the remodeling of dimers into the chemically active synaptic tetramer and the regulation of subunit rotation during DNA exchange. We identify a set of hydrophobic residues within the oligomerization helix that controls these steps by the Hin DNA invertase. Phe105 and Met109 insert into hydrophobic pockets within the catalytic domain of the same subunit to stabilize the inactive dimer conformation. These rotate out of the catalytic domain in the dimer and into the subunit rotation interface of the tetramer. About half of residue 105 and 109 substitutions gain the ability to generate stable synaptic tetramers and/or promote DNA chemistry without activation by the Fis/enhancer element. Phe106 replaces Phe105 in the catalytic domain pocket to stabilize the tetramer conformation. Significantly, many of the residue 105 and 109 substitutions support subunit rotation but impair ligation, implying a defect in rotational pausing at the tetrameric conformer poised for ligation. We propose that a ratchet-like surface involving Phe105, Met109 and Leu112 within the rotation interface functions to gate the subunit rotation reaction. Hydrophobic residues are present in analogous positions in other serine recombinases and likely perform similar functions.

Reversible site-specific DNA inversion reactions are widely distributed in bacteria and their viruses. They control a range of biological reactions that most often involve alterations of molecules on the surface of cells or phage. These programmed DNA rearrangements usually occur at a low frequency, thereby preadapting a small subset of the population to a change in environmental conditions, or in the case of phages, an expanded host range. A dedicated recombinase, sometimes with the aid of additional regulatory or DNA architectural proteins, catalyzes the inversion of DNA. RecA or other components of the general recombination-repair machinery are not involved. This chapter discusses site-specific DNA inversion reactions mediated by the serine recombinase family of enzymes and focuses on the extensively studied serine DNA invertases that are stringently controlled by the Fis-bound enhancer regulatory system. The first section summarizes biological features and general properties of inversion reactions by the Fis/enhancer-dependent serine invertases and the recently described serine DNA invertases in Bacteroides . Mechanistic studies of reactions catalyzed by the Hin and Gin invertases are then explored in more depth, particularly with regards to recent advances in our understanding of the function of the Fis/enhancer regulatory system. These include the steps leading to the formation of the active recombination complex (invertasome) containing the recombinase tetramer and Fis/enhancer element and the process of DNA strand exchange by rotation of synapsed subunit pairs within the invertasome. The role of DNA topological forces that function in concert with the Fis/enhancer controlling element in specifying the overwhelming bias for DNA inversion over deletion and intermolecular recombination is also discussed.

Efficient assembly of RAG1/2-recombination signal sequence (RSS) DNA complexes that are competent for V(D)J cleavage requires the presence of the nonspecific DNA binding and bending protein HMGB1 or HMGB2. We find that either of the two minimal DNA binding domains of HMGB1 is effective in assembling RAG1/2-RSS complexes on naked DNA and stimulating V(D)J cleavage but that both domains are required for efficient activity when the RSS is incorporated into a nucleosome. The single-domain HMGB protein from Saccharomyces cerevisiae, Nhp6A, efficiently assembles RAG1/2 complexes on naked DNA; however, these complexes are minimally competent for V(D)J cleavage. Nhp6A forms much more stable DNA complexes than HMGB1, and a variety of mutations that destabilize Nhp6A binding to bent microcircular DNA promote increased V(D)J cleavage. One of the two DNA bending wedges on Nhp6A and the analogous phenylalanine wedge at the DNA exit site of HMGB1 domain A were found to be essential for promoting RAG1/2-RSS complex formation. Because the phenylalanine wedge is required for specific recognition of DNA kinks, we propose that HMGB proteins facilitate RAG1/2-RSS interactions by recognizing a distorted DNA structure induced by RAG1/2 binding. The resulting complex must be sufficiently dynamic to enable the series of RAG1/2-mediated chemical reactions on the DNA.

Most site-specific recombinases can be grouped into two mechanistically distinct families. Whereas tyrosine recombinases exchange DNA strands through a Holliday intermediate, serine recombinases such as Hin generate double-strand breaks in each recombining partner. Here, site-directed protein crosslinking is used to elucidate the configuration of protein subunits and DNA within the Hin synaptic complex and to follow the movement of protein subunits during DNA strand exchange. Our results show that the protein interface mediating synapsis is localized to a region within the catalytic domains, thereby positioning the DNA strands on the outside of the Hin tetrameric complex. Unexpected crosslinks between residues within the dimerization helices provide evidence for a conformational change that accompanies DNA cleavage. We demonstrate that the Hin subunits, which are linked to the cleaved DNA ends by serine-phosphodiester bonds, translocate between synapsed dimers to exchange the DNA strands.

The Hin DNA invertase promotes a site-specific DNA recombination reaction in the Salmonella chromosome. The native Hin reaction exhibits overwhelming selectivity for promoting inversions between appropriately oriented recombination sites and requires the Fis regulatory protein, a recombinational enhancer, and a supercoiled DNA substrate. Here, we report a robust recombination reaction employing oligonucleotide substrates and a hyperactive mutant form of Hin. Synaptic complex intermediates purified by gel electrophoresis were found to contain four Hin protomers bound to two recombination sites. Each Hin protomer is associated covalently with a cleaved DNA end. The cleaved complexes can be ligated into both parental and recombinant orientations at equivalent frequencies, provided the core residues can base-pair, and are readily disassembled into separated DNA fragments bound by Hin dimers. Kinetic analyses reveal that synapsis occurs rapidly, followed by comparatively slow Hin-catalyzed DNA cleavage. Subsequent steps of the reaction, including DNA exchange and ligation, are fast. Thus, post-synaptic step(s) required for DNA cleavage limit the overall rate of the recombination reaction.

Summary The leuV operon of Escherichia coli encodes three of the four genes for the tRNA1Leu isoacceptors. Transcription from this and other stable RNA promoters is known to be affected by a cis-acting UP element and by Fis protein interactions with the carboxyl-terminal domain of the α-subunits of RNA polymerase. In this report, we suggest that transcription from the leuV promoter also is activated by a Fis-mediated, DNA supercoiling-dependent mechanism similar to the IHF-mediated mechanism described previously for the ilvPG promoter (S. D. Sheridan et al., 1998, J Biol Chem 273: 21298–21308). We present evidence that Fis binding results in the translocation of superhelical energy from the promoter-distal portion of a supercoiling-induced DNA duplex destabilized (SIDD) region to the promoter-proximal portion of the leuV promoter that is unwound within the open complex. A mutant Fis protein, which is defective in contacting the carboxyl-terminal domain of the α-subunits of RNA polymerase, remains competent for stimulating open complex formation, suggesting that this DNA supercoiling-dependent component of Fis-mediated activation occurs in the absence of specific protein interactions between Fis and RNA polymerase. Fis-mediated translocation of superhelical energy from upstream binding sites to the promoter region may be a general feature of Fis-mediated activation of transcription at stable RNA promoters, which often contain A+T-rich upstream sequences.

5,6-Dihydrouridine (D) is a modified base found abundantly in the D-loops of tRNA from Archaea, Bacteria, and Eukarya. D is thought to be formed post-transcriptionally by the reduction of uridines in tRNA transcripts. Despite its abundance, no enzymes that catalyze D-formation have been identified. Using comparative genomics and computational methods we have identified members of the cluster of orthologous genes, COG0042, as putative dihydrouridine synthase encoding genes. Escherichia coli contains three COG0042 family members (yjbN, yhdG, and yohI). Strains were created where one, two, or all three of the COG0042 genes were deleted. Purified tRNA samples were investigated from the three single and the three double knockout strains, as well as from the triple deletion strain. The results showed that the COG0042 gene family is responsible for tRNA-dihydrouridine synthase activity in E. coli. They also suggest that the COG0042-encoded family members act site-specifically on the tRNA D-loop and contain non-redundant catalytic functions in vivo.

HMGB1 (also called HMG-1) is a DNA-bending protein that augments the affinity of diverse regulatory proteins for their DNA sites. Previous studies have argued for a specific interaction between HMGB1 and target proteins, which leads to cooperative binding of the complex to DNA. Here we propose a different model that emerged from studying how HMGB1 stimulates enhanceosome formation by the Epstein-Barr viral activator Rta on a target gene, BHLF-1. HMGB1 stimulates binding of individual Rta dimers to multiple sites in the enhancer. DNase I and hydroxyl radical footprinting, electrophoretic mobility shift assays, and immobilized template assays failed to reveal stable binding of HMGB1 within the complex. Furthermore, mutational analysis failed to identify a specific HMGB1 target sequence. The effect of HMGB1 on Rta could be reproduced by individual HMG domains, yeast HMO1, or bacterial HU. These results, combined with the effects of single-amino-acid substitutions within the DNA-binding surface of HMGB1 domain A, argue for a mechanism whereby DNA-binding and bending by HMGB1 stimulate Rta-DNA complex formation in the absence of direct interaction with Rta or a specific HMGB1 target sequence. The data contrast with our analysis of HMGB1 action on another BHLF-1 regulatory protein called ZEBRA. We discuss the two distinct modes of HMGB1 action on a single regulatory region and propose how HMGB1 can function in diverse contexts.

Nhp6A is an abundant non-histone chromatin-associated protein in Saccharomyces cerevisiae that contains a minor groove DNA binding motif called the HMG box. In this report, we show that Nhp6Ap binds to cisplatin intrastrand cross-links on duplex DNA with a 40-fold greater affinity than to unmodified DNA with the same sequence. Nevertheless, Nhp6Ap bound to cisplatinated DNA readily exchanges onto unmodified DNA. Phenanthroline-copper footprinting and two-dimensional NMR on complexes of wild-type and mutant Nhp6Ap with DNA were employed to probe the mode of binding to the cisplatin lesion. Recognition of the cisplatin adduct requires a surface-exposed phenylalanine on Nhp6Ap that promotes bending of DNA by inserting into the helix from the minor groove. We propose that Nhp6Ap targets the cisplatin adduct by means of intercalation by the phenylalanine and that it can bind in either orientation with respect to the DNA lesion. A methionine, which also inserts between base pairs and functions in target selection on unmodified DNA, plays no apparent role in recognition of the cisplatin lesion. Basic amino acids within the N-terminal arm of Nhp6Ap are required for high-affinity binding to the cisplatin adduct as well as to unmodified DNA. Cisplatin mediates its cytotoxicity by forming covalent adducts on DNA, and we find that Deltanhp6a/b mutants are hypersensitive to cisplatin in comparison with the wild-type strain. In contrast, Deltanhp6a/b mutants are slightly more resistant to hydrogen peroxide and ultraviolet irradiation. Therefore, Nhp6A/Bp appears to directly or indirectly function in yeast to enhance cellular resistance to cisplatin.

The Hin recombinase specifically recognizes its DNA-binding site by means of both major and minor groove interactions. A previous X-ray structure, together with new structures of the Hin DNA-binding domain bound to a recombination half-site that were solved as part of the present study, have revealed that two ordered water molecules are present within the major groove interface. In this report, we test the importance of these waters directly by X-ray crystal structure analysis of complexes with four mutant DNA sequences. These structures, combined with their Hin-binding properties, provide strong support for the critical importance of one of the intermediate waters. A lesser but demonstrable role is ascribed to the second water molecule. The mutant structures also illustrate the prominent roles of thymine methyls both in stabilizing intermediate waters and in interfering with water or amino acid side chain interactions with DNA.

The Saccharomyces cerevisiae non-histone protein 6-A (NHP6A) is a member of the high-mobility group 1/2 protein family that bind and bend DNA of mixed sequence. NHP6A has only one high-mobility group 1/2 DNA binding domain and also requires a 16-amino-acid basic tail at its N-terminus for DNA binding. We show in this report that nuclear accumulation of NHP6A is strictly correlated with its DNA binding properties since only nonhistone protein 6 A-green fluorescent protein chimeras that were competent for DNA binding were localized to the nucleus. Despite the requirement for basic residues within the N-terminal segment for DNA binding and nuclear accumulation, this region does not appear to contain a nuclear localization signal. Moreover, NHP6A does not bind to the yeast nuclear localization signal receptor SRP1 and nuclear targeting of NHP6A does not require the function of the 14 different importins. Unlike histone H2B1 which contains a classical nuclear localization signal, entry of NHP6A into the nucleus was found to be independent of Ran as judged by coexpression of Ran GTPase mutants and was shown to occur at 0 degrees C after a 15-min induction. These unusual properties lead us to suggest that NHP6A entry into the nucleus proceeds by a nonclassical Ran-independent pathway.

The Escherichia coli proP P2 promoter, which directs the expression of an integral membrane transporter of proline, glycine betaine, and other osmoprotecting compounds, is induced upon entry into stationary phase to protect cells from osmotic shock. Transcription from the P2 promoter is completely dependent on RpoS (ς 38 ) and Fis. Fis activates transcription by binding to a site centered at −41, which overlaps the promoter, where it makes a specific contact with the C-terminal domain of the α subunit of RNA polymerase (α-CTD). We show here that Fis and cyclic AMP (cAMP) receptor protein (CRP)-cAMP collaborate to activate transcription synergistically in vitro. Coactivation both in vivo and in vitro is dependent on CRP binding to a site centered at −121.5, but CRP without Fis provides little activation. The contribution by CRP requires the correct helical phasing of the CRP site and a functional activation region 1 on CRP. We provide evidence that coactivation is achieved by Fis and CRP independently contacting each of the two α-CTDs. Efficient transcription in vitro requires that both activators must be preincubated with the DNA prior to addition of RNA polymerase.

Transcription of the P1 promoter of the Escherichia coli proP gene, which encodes a transporter of osmoprotectants, is strongly induced by a shift to hyperosmotic media. Unlike most other osmotically regulated promoters, the induction occurs for a brief period of time, corresponding to the replacement of intracellular K(+) glutamate with osmoprotecting compounds. This burst of proP transcription is correlated with the osmolarity-dependent binding of the cAMP receptor protein CRP to a site within the proP P1 promoter. We show that CRP-cAMP functions as an osmotically sensitive repressor of proP P1 transcription in vitro. Binding of CRP to the proP promoter in vivo is transiently destabilized after a hyperosmotic shift with kinetics that correspond to the derepression of transcription, whereas Fis and Lac repressor binding is not osmotically sensitive. Similar osmotic regulation of proP P1 transcription by the CRP* mutant implies that binding of cAMP is not responsible for the unusual osmotic sensitivity of CRP activity. Osmotic regulation of CRP activity is not limited to proP. Activation of the lac promoter by CRP is also transiently inhibited after an osmotic upshift, as is the binding of CRP to the galdelta4P1 promoter. These findings suggest that CRP functions in certain contexts to regulate gene expression in response to osmotic changes, in addition to its role in catabolite control.

In a recent issue of Molecular Cell, Mouw et al. (2008) report a crystal structure of a serine recombinase bound to a regulatory DNA site in an unexpected synaptic complex configuration, which forms the framework for a new model of the entire 12 subunit, 186 bp deletion complex.

The Hin DNA invertase becomes catalytically activated when assembled in an invertasome complex containing two Fis dimers bound to an enhancer segment. The region of Fis responsible for transactivation of Hin contains a mobile β-hairpin arm that extends from each dimer subunit. We show here that whereas both Fis dimers must be capable of activating Hin, Fis heterodimers that have only one functional activating β-arm are sufficient to form catalytically competent invertasomes. Analysis of homodimer and heterodimer mixes of different Hin mutants suggests that Fis must activate each subunit of the two Hin dimers that participate in catalysis. These experiments also indicate that all four Hin subunits must be coordinately activated prior to initiation of the first chemical step of the reaction and that the process of activation is independent of the catalytic steps of recombination. We propose a molecular model for the invertasome structure that is consistent with current information on protein–DNA structures and the topology of the DNA strands within the recombination complex. In this model, a single Fis activation arm could contact amino acids from both Hin subunits at the dimer interface to induce a conformational change that coordinately positions the active sites close to the scissile phosphodiester bonds.

The conversion from an alpha-helix to a beta-strand has received extensive attention since this structural change may induce many amyloidogenic proteins to self-assemble into fibrils and cause fatal diseases. Here we report the conversion of a peptide segment from a beta-strand to an alpha-helix by a single-site mutation as observed in the crystal structure of Fis mutant Pro26Ala determined at 2.0 A resolution. Pro26 in Fis occurs at the point where a flexible extended beta-hairpin arm leaves the core structure. Thus it can be classified as a "hinge proline" located at the C-terminal end of the beta2-strand and the N-terminal cap of the A alpha-helix. The replacement of Pro26 to alanine extends the A alpha-helix for two additional turns in one of the dimeric subunits; therefore, the structure of the peptide from residues 22 to 26 is converted from a beta-strand to an alpha-helix. This result confirms the structural importance of the proline residue located at the hinge region and may explain the mutant's reduced ability to activate Hin-catalyzed DNA inversion. The peptide (residues 20 to 26) in the second monomer subunit presumably retains its beta-strand conformation in the crystal; therefore, this peptide shows a "chameleon-like" character since it can adopt either an alpha-helix or a beta-strand structure in different environments. The structure of Pro26Ala provides an additional example where not only the protein sequence, but also non-local interactions determine the secondary structure of proteins.

The non-histone proteins 6A/B (NHP6A/B) ofSaccharomyces cerevisiae are high mobility group proteins that bind and severely bend DNA of mixed sequence. They exhibit high affinity for linear DNA and even higher affinity for microcircular DNA. The 16-amino acid basic segment located N-terminal to the high mobility group domain is required for stable complex formation on both linear and microcircular DNA. Although mutants lacking the N terminus are able to promote microcircle formation and Hin invertasome assembly at high protein concentrations, they are unable to form stable complexes with DNA, co-activate transcription, and complement the growth defect ofΔnhp6a/b mutants. A basic patch between amino acids 13 and 16 is critical for these activities, and a second basic patch between residues 8 and 10 is required for the formation of monomeric complexes with linear DNA. Mutational analysis suggests that proline 18 may direct the path of the N-terminal arm to facilitate DNA binding, whereas the conserved proline at position 21, tyrosine 28, and phenylalanine 31 function to maintain the tertiary structure of the high mobility group domain. Methionine 29, which may intercalate into DNA, is essential for NHP6A-induced microcircle formation of 75-bp but not 98-bp fragments in vitro, and for full growth complementation of Δnhp6a/b mutants in vivo.

The Fis protein regulates site-specific DNA inversion catalyzed by a family of DNA invertases when bound to a cis-acting recombinational enhancer. As is often found for transactivation domains, previous crystal structures have failed to resolve the conformation of the N-terminal inversion activation region within the Fis dimer. A new crystal form of a mutant Fis protein now reveals that the activation region contains two beta-hairpin arms that protrude over 20 A from the protein core. Saturation mutagenesis identified the regulatory and structurally important amino acids. The most critical activating residues are located near the tips of the beta-arms. Disulfide cross-linking between the beta-arms demonstrated that they are highly flexible in solution and that efficient inversion activation can occur when the beta-arms are covalently linked together. The emerging picture for this regulatory motif is that contacts with the recombinase at the tip of the mobile beta-arms activate the DNA invertase in the context of an invertasome complex.

Serine recombinases are often tightly controlled by elaborate, topologically-defined, nucleoprotein complexes. Hin is a member of the DNA invertase subclass of serine recombinases that are regulated by a remote recombinational enhancer element containing two binding sites for the protein Fis. Two Hin dimers bound to specific recombination sites associate with the Fis-bound enhancer by DNA looping where they are remodeled into a synaptic tetramer competent for DNA chemistry and exchange. Here we show that the flexible beta-hairpin arms of the Fis dimers contact the DNA binding domain of one subunit of each Hin dimer. These contacts sandwich the Hin dimers to promote remodeling into the tetramer. A basic region on the Hin catalytic domain then contacts enhancer DNA to complete assembly of the active Hin tetramer. Our results reveal how the enhancer generates the recombination complex that specifies DNA inversion and regulates DNA exchange by the subunit rotation mechanism. DOI: http://dx.doi.org/10.7554/eLife.01211.001, eLife digest Many processes in biology rely on enzymes that break both the strands in a DNA molecule, then rearrange the strands, and finally join them back together in a new configuration. These recombination reactions can, for example, change the positions of genetic elements such as enhancers and promoters within the DNA molecule and, therefore, influence how a given gene is expressed as a protein. Cells need to be able to control recombination reactions because they can lead to leukemia and lymphomas if they go wrong. The enzymes that catalyze these recombination reactions are called recombinases. One type of recombinase binds to specific sequences of DNA bases and uses an amino acid in the enzyme–usually serine or tyrosine–to break and rejoin the DNA strands. Recombination reactions require the assembly of complexes containing many proteins bound to DNA. Tyrosine recombinases form relatively simple protein-DNA complexes, and these have been studied in detail. Serine recombinases, on the other hand, form more elaborate protein-DNA complexes, and much less is known about these. Now McLean et al. have unraveled the mechanism that a serine recombinase called Hin uses to reverse the direction of a stretch of chromosomal DNA in the bacteria Salmonella enterica. Inverting this stretch of DNA–which contains about 1000 base pairs–changes the position of a gene promoter that is responsible for the production of flagellin, which is the protein that enables the bacterium to move. This is one of the tricks that Salmonella uses to evade the immune system of its host. Previous research has established that four Hin subunits and two copies of a protein called Fis are needed to invert this stretch of DNA: two Hin subunits bind to each of the two hix recombination sites, and the Fis proteins (which are dimers) bind to each end of an enhancer that is located between the hix sites. A protein called HU then causes the DNA to bend and form a loop, and the four Hin subunits and the two Fis dimers all come together at the enhancer to form a structure called the invertasome where the recombination reaction occurs. All four DNA strands at the crossover point are broken as a result of a near simultaneous attack by the catalytic serine amino acids in the Hin subunits. One pair of Hin subunits–and the two DNA strands attached to them–then rotate by 180 degrees around the other pair of Hin subunits. This means that the stretch of DNA between the hix sites is inverted when the DNA strands are rejoined at the end of the reaction. Enhancers often regulate transcription and other reactions from a distance. McLean et al. reveal how an enhancer of a DNA recombination reaction works. The pairs of Hin subunits that initially bind to the DNA are not catalytically active, but when they are brought together by the enhancer and form a tetramer, they become active. Two of the Hin subunits are clamped onto the enhancer by the Fis dimers and by directly interacting with the enhancer DNA, but the other two (and the DNA strands attached to them) are free to rotate within the tetramer. In the Salmonella chromosome the enhancer is located close to one of the hix sites (∼100 base pairs away from it), so the length of the DNA between the enhancer and hix site physically limits the number of Hin subunit rotations to just one. DOI: http://dx.doi.org/10.7554/eLife.01211.002

Background A large subfamily of serine recombinases contains long polypeptide segments appended to the C-terminal end of the conserved catalytic domain. Members of this subfamily often function as phage integrases but also mediate transposition and regulate terminal differentiation processes in eubacteria. Although a few members of this subfamily have been studied in purified in vitro systems, key mechanistic aspects of reactions promoted by these recombinases remain to be determined, particularly with respect to the functions of the large C-terminal domain. Results We have developed and characterized a robust in vitro recombination reaction by the Listeria phage A118 integrase, a member of the subfamily of serine recombinases containing a large C-terminal domain. The reaction occurs in a simple buffered salt solution and exhibits a modest stimulation by divalent cations or spermidine and DNA supercoiling. Recombination with purified A118 integrase is unidirectional, being efficient only between attP and attB DNA sites to either join separate DNA molecules (intermolecular recombination) or to generate deletions or inversions depending on the relative orientation of att sites in cis (intramolecular recombination). The minimal attP site is 50 bp but requires only 44 bp of base sequence information, whereas the minimal attB site is 42 bp and requires 38 bp of base sequence information. DNA exchange occurs between the central 2 bp of attP and attB. Identity between these two base pairs is required for recombination, and they solely determine the orientation of recombination sites. The integrase dimer binds efficiently to full att sites, including the attL and attR integration products, but poorly and differentially to each half-site. The large C-terminal domain can be separated from the N-terminal catalytic by partial proteolysis and mediates non-cooperative DNA binding to att sites. Conclusions The basic properties of the phage A118 integrase reaction and its substrate requirements have been elucidated. A118 integrase thus joins the handful of biochemically characterized serine integrases that are serving as models for mechanistic studies on this important class of recombinases. Information reported here will also be useful in exploiting this recombinase for genetic engineering.

Nonhistone proteins 6A and 6B (NHP6A/B) are nonsequence-specific DNA-binding proteins from Saccharomyces cerevisiae that are related structurally and functionally to the mammalian high mobility group proteins 1 and 2. These DNA architectural proteins distort DNA structure severely and have been shown to promote assembly of specialized recombination complexes. Here we show that the yeast NHP6A/B proteins are required for the induction of a subset of genes transcribed by RNA polymerase II (pol II). Activation of the CUP1, CYC1, GAL1, and DDR2 genes was decreased or abolished completely in the delta nhp6A/B strain. No significant change in basal expression was observed for any of the 10 genes examined. Analysis of chimeric gene constructs localized the regions dependent on NHP6A/B to be primarily at the core promoters, although the GAL1 UAS also requires NHP6A/B for activity. In vitro, NHP6A stimulated transcription by pol II at the GAL1 promoter three- to fivefold above the level of activation by GAL4-VP16 alone. Gel mobility shift assays showed that NHP6A promotes the formation of a complex with TBP and TFIIA at the TATA box that has enhanced affinity for TFIIB.

The formation of higher order protein˙DNA structures often requires bending of DNA strands between specific sites, a process that can be facilitated by the action of nonspecific DNA-binding proteins which serve as assembly factors. A model for this activity is the formation of the invertasome, an intermediate structure created in the Hin-mediated site-specific DNA inversion reaction, which is stimulated by the prokaryotic nucleoid-associated protein HU. Previously, we have shown that the mammalian HMG1/2 proteins substitute for HU in this system and display efficient DNA wrapping activity in vitro. In the present work, we isolate the primary sources of assembly factor activity in Saccharomyces cerevisiae, as measured by the ability to stimulate invertasome formation, and show that these are the previously identified NHP6A/B proteins. NHP6A/B have comparable or greater activity in DNA binding, bending, and supercoiling with respect to HU and HMG1 and appear to form more stable protein˙DNA complexes. In addition, expression of NHP6A in mutant Escherichia coli cells lacking HU and Fis restores normal morphological appearance to these cells, specifically in nucleoid condensation and segregation. From these data we predict diverse architectural roles for NHP6A/B in manipulating chromosome structure and promoting the assembly of multicomponent protein˙DNA complexes.

The fis operon from Salmonella typhimurium has been cloned and sequenced, and the properties of Fis-deficient and Fis-constitutive strains were examined. The overall fis operon organization in S. typhimurium is the same as that in Escherichia coli, with the deduced Fis amino acid sequences being identical between both species. While the open reading frames upstream of fis have diverged slightly, the promoter regions between the two species are also identical between -49 and +94. Fis protein and mRNA levels fluctuated dramatically during the course of growth in batch cultures, peaking at approximately 40,000 dimers per cell in early exponential phase, and were undetectable after growth in stationary phase. fis autoregulation was less effective in S. typhimurium than that in E. coli, which can be correlated with the absence or reduced affinity of several Fis-binding sites in the S. typhimurium fis promoter region. Phenotypes of fis mutants include loss of Hin-mediated DNA inversion, cell filamentation, reduced growth rates in rich medium, and increased lag times when the mutants are subcultured after prolonged growth in stationary phase. On the other hand, cells constitutively expressing Fis exhibited normal logarithmic growth but showed a sharp reduction in survival during stationary phase. During the course of these studies, the sigma 28-dependent promoter within the hin-invertible segment that is responsible for fljB (H2) flagellin synthesis was precisely located.

We examine whether the Escherichia coli chromosome is folded into a self-adherent nucleoprotein complex, or alternately is a confined but otherwise unconstrained self-avoiding polymer. We address this through in vivo visualization, using an inducible GFP fusion to the nucleoid-associated protein Fis to non-specifically decorate the entire chromosome. For a range of different growth conditions, the chromosome is a compact structure that does not fill the volume of the cell, and which moves from the new pole to the cell centre. During rapid growth, chromosome segregation occurs well before cell division, with daughter chromosomes coupled by a thin inter-daughter filament before complete segregation, whereas during slow growth chromosomes stay adjacent until cell division occurs. Image correlation analysis indicates that sub-nucleoid structure is stable on a 1 min timescale, comparable to the timescale for redistribution time measured for GFP-Fis after photobleaching. Optical deconvolution and writhe calculation analysis indicate that the nucleoid has a large-scale coiled organization rather than being an amorphous mass. Our observations are consistent with the chromosome having a self-adherent filament organization.