The role of the chromatin-associated protein Hbsu in β-mediated DNA recombination is to facilitate the joining of distant recombination sites

The β recombinase is unable to mediate in vitro DNA recombination between two directly oriented recombination sites unless a bacterial chromatin-associated protein ( Bacillus subtilis Hbsu or Eschrichia coli HU) is provided. By electron microscopy, we show that the role of Hbsu is to help in joining the recombination sites to form a stable synaptic complex. Some evidence supports the fact that Hbsu works by recognizing and stabilizing a DNA structure at the recombination site, rather than by serving as a bridge between β recombinase dimers through a protein-protein interaction. We show that the mammalian HMG1 protein, which shares neither sequence nor structural homology with Hbsu, can also stimulate β-mediated recombination. These chromatin-associated proteins share the property of binding to DNA in a relatively non-specific fashion, bending it, and having a marked preference for altered DNA structures. Hbsu, HU or HMG1 proteins probably bind specifically at the crossing-over region, since at limiting protein-DNA molar ratios they could not be outcompeted by an excess of a DNA lacking the crossing over site. Distamycin, a minor groove binder that induces local distortions in DNA, did not affect the binding of β protein to DNA, but inhibited the formation of the synaptic complex.     

Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation

Catalysis of DNA recombination by Tn3 resolvase is conditional on prior formation of a synapse, comprising 12 resolvase subunits and two recombination sites (res). Each res binds a resolvase dimer at site I, where strand exchange takes place, and additional dimers at two adjacent 'accessory' binding sites II and III. 'Hyperactive' resolvase mutants, that catalyse strand exchange at site I without accessory sites, were selected in E. coli. Some single mutants can resolve a res x site I plasmid (that is, with one res and one site I), but two or more activating mutations are necessary for efficient resolution of a site I x site I plasmid. Site I x site I resolution by hyperactive mutants can be further stimulated by mutations at the crystallographic 2-3' interface that abolish activity of wild-type resolvase. Activating mutations may allow regulatory mechanisms of the wild-type system to be bypassed, by stabilizing or destabilizing interfaces within and between subunits in the synapse. The positions and characteristics of the mutations support a mechanism for strand exchange by serine recombinases in which the DNA is on the outside of a recombinase tetramer, and the tertiary/quaternary structure of the tetramer is reconfigured.     

Transposon-mediated site-specific recombination in vitro: DNA cleavage and protein-DNA linkage at the recombination site

Resolvase, the product of the tnpR gene of the transposable element gamma delta, mediates a site-specific recombination between two copies of the element directly repeated on the same replicon. The resolution site, res, at which resolvase acts lies in the intercistronic region between the tnpA and tnpR genes. We have studied this site-specific recombination in vitro. In the absence of Mg2+, a resolvase-res complex is formed, which contains DNA molecules that have been cleaved at res. Our data suggest that in this complex resolvase is covalently attached to the 5' ends of the cleaved DNA, leaving free 3' hydroxyl groups. DNA cleavage is stimulated by the interaction of two res sites on the same substrate molecule and appears to be an intermediate step in normal res site recombination. We show that the DNA is cut within a region previously identified as containing the crossover point at the palindromic sequence 5'- (see formula in text) to generate 3' extensions of two bases.     

Contacts between gamma delta resolvase and the gamma delta res site.

We have investigated the interaction between resolvase and the res site of the transposon gamma delta by methylation and ethylation interference experiments. We have examined the effect of these DNA modifications both on binding and resolution in vitro. Major groove methylations within a 9 bp sequence that borders each site inhibit binding of resolvase to that site. Ethylation of certain phosphates within, and adjacent to, this border sequence inhibits binding. Together, these interference points define a contact region, present at all three res sites. In vitro resolution is inhibited only by modifications within site I. Inhibition of resolution by methylation of adenines at the center of site I suggests that minor groove contacts near the crossover may be required for resolution activity.     

Replication terminus for DNA polymerase I during initiation of pAMβ1 replication: role of the plasmid-encoded resolution system

Replication of plasmid pAMβ1 is initiated by DNA polymerase I (Pol I) and completed by DNA polymerase III holoenzyme contained in the replisome machinery. In this study we report that initiation of DNA replication generates D-loop structures containing the nascent leading strand paired to its template, and that D-loop extension is arrested ≈230 bp from the initiation site of DNA synthesis in the presence of the plasmid-encoded resolvase. In vitro and in vivo data suggest that this arrest is caused by a collision between Pol I and the resolvase bound to its target. As the arrested D-loop replication intermediates carry a single-stranded primosome-assembly site, we hypothesize that the biological role of the replication arrest is to limit the region replicated by Pol I and to promote the replacement of Pol I by the replisome in order to initiate concerted synthesis of the leading and lagging strands.     

Synapsis and catalysis by activated Tn3 resolvase mutants

The serine recombinase Tn3 resolvase catalyses recombination between two 114 bp res sites, each of which contains binding sites for three resolvase dimers. We have analysed the in vitro properties of resolvase variants with ‘activating’ mutations, which can catalyse recombination at binding site I of res when the rest of res is absent. Site I × site I recombination promoted by these variants can be as fast as res × res recombination promoted by wild-type resolvase. Activated variants have reduced topological selectivity and no longer require the 2–3′ interface between subunits that is essential for wild-type resolvase-mediated recombination. They also promote formation of a stable synapse comprising a resolvase tetramer and two copies of site I. Cleavage of the DNA strands by the activated mutants is slow relative to the rate of synapsis. Stable resolvase tetramers were not detected in the absence of DNA or bound to a single site I. Our results lead us to conclude that the synapse is assembled by sequential binding of resolvase monomers to site I followed by interaction of two site I-dimer complexes. We discuss the implications of our results for the mechanisms of synapsis and regulation in recombination by wild-type resolvase.     

Identification of a multimer resolution system involved in stabilization of the Salmonella dublin virulence plasmid pSDL2.

The Salmonella dublin virulence plasmid pSDL2 is a low-copy-number plasmid that is highly conserved in its host. Deletion of the 8-kb EcoRI C fragment downstream of the virulence region leads to plasmid instability and formation of multimers. We identified a multimer resolution system in the EcoRI C fragment composed of a trans-acting resolvase gene and a cis-acting resolution site. The resolvase gene, rsd, maps within a 2-kb EcoRV fragment and appears to be part of a multicistronic unit together with at least two other genes of unknown function. The derived protein, 28.7-kDa in size, is almost identical to the D protein of miniF. The C-terminal region was shown to have substantial similarity to the conserved C-terminal domains of the site-specific recombinases of the integrase family. The cis-acting resolution site, crs, is located upstream of rsd within a 628-bp SmaI-HpaI fragment. It contains eight direct incomplete 17-bp repeats followed by a segment rich in indirect repeats, the latter being homologous to the oriV1 sequence of miniF. crs contains the crossover site for specific recombination and mediates bidirectional promoter activity. A replicative function in analogy to that of oriV1 of F could not be demonstrated. The multimer resolution system was shown to stabilize pACYC184 and is dependent on the recA-mediated formation of multimeric plasmids. Screening different Salmonella serovars with a pSDL2-specific recombination assay revealed that only strains harboring a virulence plasmid encode for resolvase activity. Our results suggest that site-specific recombination contributes to the stable inheritance of pSDL2 and other Salmonella virulence plasmids.     

The first structure of an RNA m5C methyltransferase, Fmu, provides insight into catalytic mechanism and specific binding of RNA substrate

The crystal structure of E. coli Fmu, determined at 1.65 A resolution for the apoenzyme and 2.1 A resolution in complex with AdoMet, is the first representative of the 5-methylcytosine RNA methyltransferase family that includes the human nucleolar proliferation-associated protein p120. Fmu contains three subdomains which share structural homology to DNA m(5)C methyltransferases and two RNA binding protein families. In the binary complex, the AdoMet cofactor is positioned within the active site near a novel arrangement of two conserved cysteines that function in cytosine methylation. The site is surrounded by a positively charged cleft large enough to bind its unique target stem loop within 16S rRNA. Docking of this stem loop RNA into the structure followed by molecular mechanics shows that the Fmu structure is consistent with binding to the folded RNA substrate.     

Site-specific relaxation and recombination by the Tn3 resolvase: recognition of the DNA path between oriented res sites

We studied the dynamics of site-specific recombination by the resolvase encoded by the Escherichia coli transposon Tn3. The pure enzyme recombined supercoiled plasmids containing two directly repeated recombination sites, called res sites. Resolvase is the first strictly site-specific topoisomerase. It relaxed only plasmids containing directly repeated res sites; substrates with zero, one or two inverted sites were inert. Even when the proximity of res sites was ensured by catenation of plasmids with a single site, neither relaxation nor recombination occurred. The two circular products of recombination were catenanes interlinked only once. These properties of resolvase require that the path of the DNA between res sites be clearly defined and that strand exchange occur with a unique geometry. A model in which one subunit of a dimeric resolvase is bound at one res site, while the other searches along adjacent DNA until it encounters the second site, would account for the ability of resolvase to distinguish intramolecular from intermolecular sites, to sense the relative orientation of sites and to produce singly interlinked catenanes. Because resolvase is a type 1 topoisomerase, we infer that it makes the required duplex bDNA breaks of recombination one strand at a time.     

Behavior of Tn3 resolvase in solution and its interaction with res

The solution properties of Tn3 resolvase (Tn3R) were studied by sedimentation equilibrium, sedimentation velocity analytical ultracentrifugation, and small-angle neutron scattering. Tn3R was found to be in a monomer-dimer self-association equilibrium, with a dissociation constant of K(D)(1-2)=50 microM. Sedimentation velocity and small-angle neutron scattering data are consistent with a solution structure of dimeric Tn3R similar to that of gammadelta resolvase in a co-crystal structure, but with the DNA-binding domains in a more extended conformation. The solution conformations of sites I, II, and III were studied with small angle x-ray scattering and modeled using rigid-body and ab initio techniques. The structures of these sites do not show any distortion, at low resolution, from B-DNA. The equilibrium binding properties of Tn3R to the individual binding sites in res were investigated by employing fluorescence anisotropy measurements. It was found that site II and site III have the highest affinity for Tn3R, followed by site I. Finally, the affinity of Tn3R for nonspecific DNA was assayed by competition experiments.     

The catalytic residues of Tn3 resolvase

To characterize the residues that participate in the catalysis of DNA cleavage and rejoining by the site-specific recombinase Tn3 resolvase, we mutated conserved polar or charged residues in the catalytic domain of an activated resolvase variant. We analysed the effects of mutations at 14 residues on proficiency in binding to the recombination site (‘site I’), formation of a synaptic complex between two site Is, DNA cleavage and recombination. Mutations of Y6, R8, S10, D36, R68 and R71 resulted in greatly reduced cleavage and recombination activity, suggesting crucial roles of these six residues in catalysis, whereas mutations of the other residues had less dramatic effects. No mutations strongly inhibited binding of resolvase to site I, but several caused conspicuous changes in the yield or stability of the synapse of two site Is observed by non-denaturing gel electrophoresis. The involvement of some residues in both synapsis and catalysis suggests that they contribute to a regulatory mechanism, in which engagement of catalytic residues with the substrate is coupled to correct assembly of the synapse.     

Functional dissection of the Schizosaccharomyces pombe Holliday junction resolvase Ydc2: in vivo role in mitochondrial DNA maintenance.

The crystal structure of the Schizosaccharomyces pombe Holliday junction resolvase Ydc2 revealed significant structural homology with the Escherichia coli resolvase RuvC but Ydc2 contains a small triple helical bundle that has no equivalent in RuvC. Two of the alpha-helices that form this bundle show homology to a putative DNA-binding motif known as SAP. To investigate the biochemical function of the triple-helix domain, truncated Ydc2 mutants were expressed in E. coli and in fission yeast. Although the truncated proteins retained all amino-acid residues that map to the structural core of RuvC including the catalytic site, deletion of the SAP motif alone or the whole triple-helix domain of Ydc2 resulted in the complete loss of resolvase activity and impaired significantly the binding of Ydc2 to synthetic junctions in vitro. These results are in full agreement with our proposal for a DNA-binding role of the triple-helix motif [Ceschini et al. (2001) EMBO J. 20, 6601-6611]. The biological effect of Ydc2 on mtDNA in yeast was probed using wild-type and several Ydc2 mutants expressed in Deltaydc2 S. pombe. The truncated mutants were shown to localize exclusively to yeast mitochondria ruling out a possible role of the helical bundle in mitochondrial targeting. Cells that lacked Ydc2 showed a significant depletion of mtDNA content. Plasmids expressing full-length Ydc2 but not the truncated or catalytically inactive Ydc2 mutants could rescue the mtDNA 'phenotype'. These results provide evidence that the Holliday junction resolvase activity of Ydc2 is required for mtDNA transmission and affects mtDNA content in S. pombe.     

Analysis of inhibitors of the site-specific recombination reaction mediated by Tn3 resolvase

The Tn3-encoded resolvase protein promotes a site-specific recombination reaction between two directly repeated copies of the recombination site res. Several inhibitors that block this event in vitro have been isolated. In this study four of these inhibitors were tested on various steps in the recombination reaction. Two inhibitors. A9387 and A1062, inhibit resolvase binding to the res site. Further, DNase I footprinting revealed that at certain concentrations of A9387 and A1062, resolvase was preferentially bound to site I of res, the site containing the recombinational crossover point. The two other inhibitors, A20812 and A21960, do not affect resolvase binding and bending of the DNA but inhibit synapse formation between resolvase and two directly repeated res sites.     

Mutants of the gamma delta resolvase: a genetic analysis of the recombination function

The resolvase protein encoded by the gamma delta transposon has two functions. It catalyzes a site-specific recombination, and it negatively regulates the expression of two transposon genes. Both functions involve the action of resolvase at the res site. To define regions of resolvase that are involved specifically in the recombination reaction, we have isolated and characterized mutants that are defective in cointegrate resolution but retain the ability to bind to res (as measured by regulatory activity). Nine independent mutants were found to contain six different amino acid substitutions among just four distinct residues. The altered residues all lie within the 140 amino acid amino-terminal domain of resolvase and fall within two clusters of amino acids that are highly conserved in other related recombinases. The regulatory properties of the mutants suggest that one of these clusters may be involved in the interaction of the catalytic domain with the crossover site.     

Mechanism of assembly of the Bis(Molybdopterin guanine dinucleotide)molybdenum cofactor in Rhodobacter sphaeroides dimethyl sulfoxide reductase

A fully defined in vitro system has been developed for studying the mechanism of assembly of the bis(molybdopterin guanine dinucleotide)molybdenum cofactor in Rhodobacter sphaeroides dimethyl sulfoxide reductase (DMSOR). R. sphaeroides DMSOR expressed in a mobA(-) Escherichia coli strain lacks molybdopterin and molybdenum but contains a full complement of guanine in the form of GMP and GDP. Escherichia coli MobA, molybdopterin-Mo, GTP, and MgCl(2) are required and sufficient for the in vitro activation of purified DMSOR expressed in the absence of MobA. High levels of MobA inhibit the in vitro activation. A chaperone is not required for the in vitro activation process. The reconstituted DMSOR can exhibit up to 73% of the activity observed in recombinant DMSOR purified from a wild-type strain. The use of radiolabeled GTP has demonstrated incorporation of the guanine moiety from the GTP into the activated DMSOR. No role was observed for E. coli MobB in the in vitro activation of apo-DMSOR. This work also represents the first time that the MobA-mediated conversion of molybdopterin to molybdopterin guanine dinucleotide has been demonstrated directly without using the activation of a molybdoenzyme as an indicator for cofactor formation.     

Mutants of Tn3 resolvase which do not require accessory binding sites for recombination activity

Tn3 resolvase promotes site-specific recombination between two res sites, each of which has three resolvase dimer-binding sites. Catalysis of DNA-strand cleavage and rejoining occurs at binding site I, but binding sites II and III are required for recombination. We used an in vivo screen to detect resolvase mutants that were active on res sites with binding sites II and III deleted (that is, only site I remaining). Mutations of amino acids Asp102 (D102) or Met103 (M103) were sufficient to permit catalysis of recombination between site I and a full res, but not between two copies of site I. A double mutant resolvase, with a D102Y mutation and an additional activating mutation at Glu124 (E124Q), recombined substrates containing only two copies of site I, in vivo and in vitro. In these novel site Ixsite I reactions, product topology is no longer restricted to the normal simple catenane, indicating synapsis by random collision. Furthermore, the mutants have lost the normal specificity for directly repeated sites and supercoiled substrates; that is, they promote recombination between pairs of res sites in linear molecules, or in inverted repeat in a supercoiled molecule, or in separate molecules.     

Activation of RuvC Holliday junction resolvase in vitro.

The Escherichia coli RuvC protein is an endonuclease that resolves Holliday junctions. In vitro, the protein shows efficient structure-specific binding of Holliday junctions, yet the rate of junction resolution is remarkably low. We have mapped the sites of cleavage on a synthetic junction through which a crossover can branch migrate through 26 bp and find that > or = 90% of the junctions were cleaved at one site. This observation of sequence-specific cleavage suggests that inefficient resolution may be due to DNA binding events which occur away from the cleavage site and are therefore non-productive. Holliday junction resolution by RuvC protein can be stimulated by a number of factors including: (i) the presence of Mn2+ (rather than Mg2+) as the divalent metal cofactor, (ii) alkaline pH (< or = 10), and (iii) elevated temperature. These observations may indicate that other proteins are required for efficient RuvC-mediated resolution.     

Rearrangements in the staphylococcal β-lactamase-encoding plasmid, plP1066, including a DNA inversion that generates two alternative transposons

The plasmid plP1066, harboured by a methicillin-resistant Staphylococcus aureus strain isolated in France, carries genes specifying β-lactamase. This plasmid undergoes numerous rearrangements. One of these was an insertion, between the genes binR and sin encoding resolvases, of a 16 kb element which displayed the characteristic features of a transposon. This putative transposon, named Tn 5404 , carried genes encoding proteins involved in its transposition, as well as a resolution system, which were indistinguishable from those of the S. aureus transposon Tn 552 . These were: p480 encoding a probable transposase, p271 encoding a putative ATP-binding protein, binL encoding a resolvase, and a resolution site, resL . In addition, Tn 5404 carried aminoglycoside-resistance genes ( aphA, str ) and the insertion sequence IS 1181 . Tn 5404 contained at its termini 116 bp imperfect inverted repeats, similar to those of Tn 552 , and was flanked by 6 bp direct repeats. Insertion of Tn 5404 close to resR and to the structural and regulatory β-lactamase genes ( blaZ, blal, blaR1 ) of plP1066, generated a 3.5 kb invertible segment flanked by inversely repeated resolution sites ( resR, resL ). This invertible segment, which carried p480 , p271 and binL , generated Tn 552 or Tn 5404 , depending on its orientation. Thus, these two transposons share their transposition and resolution systems.     

In vivo assay for low-activity mutant forms of Escherichia coli ribonucleotide reductase

Ribonucleotide reductase (RNR) catalyzes the essential production of deoxyribonucleotides in all living cells. In this study we have established a sensitive in vivo assay to study the activity of RNR in aerobic Escherichia coli cells. The method is based on the complementation of a chromosomally encoded nonfunctional RNR with plasmid-encoded RNR. This assay can be used to determine in vivo activity of RNR mutants with activities beyond the detection limits of traditional in vitro assays. E. coli RNR is composed of two homodimeric proteins, R1 and R2. The R2 protein contains a stable tyrosyl radical essential for the catalysis that takes place at the R1 active site. The three-dimensional structures of both proteins, phylogenetic studies, and site-directed mutagenesis experiments show that the radical is transferred from the R2 protein to the active site in the R1 protein via a radical transfer pathway composed of at least nine conserved amino acid residues. Using the new assay we determined the in vivo activity of mutants affecting the radical transfer pathway in RNR and identified some residual radical transfer activity in two mutant R2 constructs (D237N and W48Y) that had previously been classified as negative for enzyme activity. In addition, we show that the R2 mutant Y356W is completely inactive, in sharp contrast to what has previously been observed for the corresponding mutation in the mouse R2 enzyme.