The selfish yeast plasmid exploits a SWI/SNF-type chromatin remodeling complex for hitchhiking on chromosomes and ensuring high-fidelity propagation.

Extra-chromosomal selfish DNA elements can evade the risk of being lost at every generation by behaving as chromosome appendages, thereby ensuring high fidelity segregation and stable persistence in host cell populations. The yeast 2-micron plasmid and episomes of the mammalian gammaherpes and papilloma viruses that tether to chromosomes and segregate by hitchhiking on them exemplify this strategy. We document for the first time the utilization of a SWI/SNF-type chromatin remodeling complex as a conduit for chromosome association by a selfish element. One principal mechanism for chromosome tethering by the 2-micron plasmid is the bridging interaction of the plasmid partitioning proteins (Rep1 and Rep2) with the yeast RSC2 complex and the plasmid partitioning locus STB. We substantiate this model by multiple lines of evidence derived from genomics, cell biology and interaction analyses. We describe a Rep-STB bypass system in which a plasmid engineered to non-covalently associate with the RSC complex mimics segregation by chromosome hitchhiking. Given the ubiquitous prevalence of SWI/SNF family chromatin remodeling complexes among eukaryotes, it is likely that the 2-micron plasmid paradigm or analogous ones will be encountered among other eukaryotic selfish elements.

The selfish yeast plasmid utilizes the condensin complex and condensed chromatin for faithful partitioning.

Equipartitioning by chromosome association and copy number correction by DNA amplification are at the heart of the evolutionary success of the selfish yeast 2-micron plasmid. The present analysis reveals frequent plasmid presence near telomeres (TELs) and centromeres (CENs) in mitotic cells, with a preference towards the former. Inactivation of Cdc14 causes plasmid missegregation, which is correlated to the non-disjunction of TELs (and of rDNA) under this condition. Induced missegregation of chromosome XII, one of the largest yeast chromosomes which harbors the rDNA array and is highly dependent on the condensin complex for proper disjunction, increases 2-micron plasmid missegregation. This is not the case when chromosome III, one of the smallest chromosomes, is forced to missegregate. Plasmid stability decreases when the condensin subunit Brn1 is inactivated. Brn1 is recruited to the plasmid partitioning locus (STB) with the assistance of the plasmid-coded partitioning proteins Rep1 and Rep2. Furthermore, in a dihybrid assay, Brn1 interacts with Rep1-Rep2. Taken together, these findings support a role for condensin and/or condensed chromatin in 2-micron plasmid propagation. They suggest that condensed chromosome loci are among favored sites utilized by the plasmid for its chromosome-associated segregation. By homing to condensed/quiescent chromosome locales, and not over-perturbing genome homeostasis, the plasmid may minimize fitness conflicts with its host. Analogous persistence strategies may be utilized by other extrachromosomal selfish genomes, for example, episomes of mammalian viruses that hitchhike on host chromosomes for their stable maintenance.

A bipartite thermodynamic-kinetic contribution by an activating mutation to RDF-independent excision by a phage serine integrase.

Streptomyces phage ϕC31 integrase (Int)-a large serine site-specific recombinase-is autonomous for phage integration (attP x attB recombination) but is dependent on the phage coded gp3, a recombination directionality factor (RDF), for prophage excision (attL x attR recombination). A previously described activating mutation, E449K, induces Int to perform attL x attR recombination in the absence of gp3, albeit with lower efficiency. E449K has no adverse effect on the competence of Int for attP x attB recombination. Int(E449K) resembles Int in gp3 mediated stimulation of attL x attR recombination and inhibition of attP x attB recombination. Using single-molecule analyses, we examined the mechanism by which E449K activates Int for gp3-independent attL x attR recombination. The contribution of E449K is both thermodynamic and kinetic. First, the mutation modulates the relative abundance of Int bound attL-attR site complexes, favoring pre-synaptic (PS) complexes over non-productively bound complexes. Roughly half of the synaptic complexes formed from Int(E449K) pre-synaptic complexes are recombination competent. By contrast, Int yields only inactive synapses. Second, E449K accelerates the dissociation of non-productively bound complexes and inactive synaptic complexes formed by Int. The extra opportunities afforded to Int(E499K) in reattempting synapse formation enhances the probability of success at fruitful synapsis.

A Flp-SUMO hybrid recombinase reveals multi-layered copy number control of a selfish DNA element through post-translational modification.

Mechanisms for highly efficient chromosome-associated equal segregation, and for maintenance of steady state copy number, are at the heart of the evolutionary success of the 2-micron plasmid as a stable multi-copy extra-chromosomal selfish DNA element present in the yeast nucleus. The Flp site-specific recombination system housed by the plasmid, which is central to plasmid copy number maintenance, is regulated at multiple levels. Transcription of the FLP gene is fine-tuned by the repressor function of the plasmid-coded partitioning proteins Rep1 and Rep2 and their antagonist Raf1, which is also plasmid-coded. In addition, the Flp protein is regulated by the host's post-translational modification machinery. Utilizing a Flp-SUMO fusion protein, which functionally mimics naturally sumoylated Flp, we demonstrate that the modification signals ubiquitination of Flp, followed by its proteasome-mediated degradation. Furthermore, reduced binding affinity and cooperativity of the modified Flp decrease its association with the plasmid FRT (Flp recombination target) sites, and/or increase its dissociation from them. The resulting attenuation of strand cleavage and recombination events safeguards against runaway increase in plasmid copy number, which is deleterious to the host-and indirectly-to the plasmid. These results have broader relevance to potential mechanisms by which selfish genomes minimize fitness conflicts with host genomes by holding in check the extra genetic load they pose.

Hitchhiking on chromosomes: A persistence strategy shared by diverse selfish DNA elements.

An underlying theme in the segregation of low-copy bacterial plasmids is the assembly of a 'segrosome' by DNA-protein and protein-protein interactions, followed by energy-driven directed movement. Analogous partitioning mechanisms drive the segregation of host chromosomes as well. Eukaryotic extra-chromosomal elements, exemplified by budding yeast plasmids and episomes of certain mammalian viruses, harbor partitioning systems that promote their physical association with chromosomes. In doing so, they indirectly take advantage of the spindle force that directs chromosome movement to opposite cell poles. Molecular-genetic, biochemical and cell biological studies have revealed several unsuspected aspects of 'chromosome hitchhiking' by the yeast 2-micron plasmid, including the ability of plasmid sisters to associate symmetrically with sister chromatids. As a result, the plasmid overcomes the 'mother bias' experienced by plasmids lacking a partitioning system, and elevates itself to near chromosome status in equal segregation. Chromosome association for stable propagation, without direct energy expenditure, may also be utilized by a small minority of bacterial plasmids-at least one case has been reported. Given the near perfect accuracy of chromosome segregation, it is not surprising that elements residing in evolutionarily distant host organisms have converged upon the common strategy of gaining passage to daughter cells as passengers on chromosomes.

Single-molecule analysis of ϕC31 integrase-mediated site-specific recombination by tethered particle motion.

Serine and tyrosine site-specific recombinases (SRs and YRs, respectively) provide templates for understanding the chemical mechanisms and conformational dynamics of strand cleavage/exchange between DNA partners. Current evidence suggests a rather intriguing mechanism for serine recombination, in which one half of the cleaved synaptic complex undergoes a 180° rotation relative to the other. The 'small' and 'large' SRs contain a compact amino-terminal catalytic domain, but differ conspicuously in their carboxyl-terminal domains. So far, only one serine recombinase has been analyzed using single substrate molecules. We now utilized single-molecule tethered particle motion (TPM) to follow step-by-step recombination catalyzed by a large SR, phage ϕC31 integrase. The integrase promotes unidirectional DNA exchange between attB and attP sites to integrate the phage genome into the host chromosome. The recombination directionality factor (RDF; ϕC31 gp3) activates the excision reaction (attL × attR). From integrase-induced changes in TPM in the presence or absence of gp3, we delineated the individual steps of recombination and their kinetic features. The gp3 protein appears to regulate recombination directionality by selectively promoting or excluding active conformations of the synapse formed by specific att site partners. Our results support a 'gated rotation' of the synaptic complex between DNA cleavage and joining.

Replication-dependent and independent mechanisms for the chromosome-coupled persistence of a selfish genome.

The yeast 2-micron plasmid epitomizes the evolutionary optimization of selfish extra-chromosomal genomes for stable persistence without jeopardizing their hosts' fitness. Analyses of fluorescence-tagged single-copy reporter plasmids and/or the plasmid partitioning proteins in native and non-native hosts reveal chromosome-hitchhiking as the likely means for plasmid segregation. The contribution of the partitioning system to equal segregation is bipartite- replication-independent and replication-dependent. The former nearly eliminates 'mother bias' (preferential plasmid retention in the mother cell) according to binomial distribution, thus limiting equal segregation of a plasmid pair to 50%. The latter enhances equal segregation of plasmid sisters beyond this level, elevating the plasmid close to chromosome status. Host factors involved in plasmid partitioning can be functionally separated by their participation in the replication-independent and/or replication-dependent steps. In the hitchhiking model, random tethering of a pair of plasmids to chromosomes signifies the replication-independent component of segregation; the symmetric tethering of plasmid sisters to sister chromatids embodies the replication-dependent component. The 2-micron circle broadly resembles the episomes of certain mammalian viruses in its chromosome-associated propagation. This unifying feature among otherwise widely differing selfish genomes suggests their evolutionary convergence to the common logic of exploiting, albeit via distinct molecular mechanisms, host chromosome segregation machineries for self-preservation.

Single molecule TPM analysis of the catalytic pentad mutants of Cre and Flp site-specific recombinases: contributions of the pentad residues to the pre-chemical steps of recombination.

Cre and Flp site-specific recombinase variants harboring point mutations at their conserved catalytic pentad positions were characterized using single molecule tethered particle motion (TPM) analysis. The findings reveal contributions of these amino acids to the pre-chemical steps of recombination. They suggest functional differences between positionally conserved residues in how they influence recombinase-target site association and formation of 'non-productive', 'pre-synaptic' and 'synaptic' complexes. The most striking difference between the two systems is noted for the single conserved lysine. The pentad residues in Cre enhance commitment to recombination by kinetically favoring the formation of pre-synaptic complexes. These residues in Flp serve a similar function by promoting Flp binding to target sites, reducing non-productive binding and/or enhancing the rate of assembly of synaptic complexes. Kinetic comparisons between Cre and Flp, and between their derivatives lacking the tyrosine nucleophile, are consistent with a stronger commitment to recombination in the Flp system. The effect of target site orientation (head-to-head or head-to-tail) on the TPM behavior of synapsed DNA molecules supports the selection of anti-parallel target site alignment prior to the chemical steps. The integrity of the synapse, whose establishment/stability is fostered by strand cleavage in the case of Flp but not Cre, appears to be compromised by the pentad mutations.

Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions.

Hexapeptides that inhibit processing of branched DNA structures induce a dynamic ensemble of Holliday junction conformations.

Holliday junctions are critical intermediates in DNA recombination, repair, and restart of blocked replication. Hexapeptides have been identified that bind to junctions and inhibit various junction-processing enzymes, and these peptides confer anti-microbial and anti-tumor properties. Earlier studies suggested that inhibition results from stabilization of peptide-bound Holliday junctions in the square planar conformation. Here, we use single molecule fluorescence resonance energy transfer (smFRET) and two model junctions, which are AT- or GC-rich at the branch points, to show that binding of the peptide KWWCRW induces a dynamic ensemble of junction conformations that differs from both the square planar and stacked X conformations. The specific features of the conformational distributions differ for the two peptide-bound junctions, but both junctions display greatly decreased Mg(2+) dependence and increased conformational fluctuations. The smFRET results, complemented by gel mobility shift and small angle x-ray scattering analyses, reveal structural effects of peptides and highlight the sensitivity of smFRET for analyzing complex mixtures of DNA structures. The peptide-induced conformational dynamics suggest multiple stacking arrangements of aromatic amino acids with the nucleobases at the junction core. This conformational heterogeneity may inhibit DNA processing by increasing the population of inactive junction conformations, thereby preventing the binding of processing enzymes and/or resulting in their premature dissociation.

Co-segregation of yeast plasmid sisters under monopolin-directed mitosis suggests association of plasmid sisters with sister chromatids.

The 2-micron plasmid, a high copy extrachromosomal element in Saccharomyces cerevisiae, propagates itself with nearly the same stability as the chromosomes of its host. Plasmid stability is conferred by a partitioning system consisting of the plasmid-coded proteins Rep1 and Rep2 and a cis-acting locus STB. Circumstantial evidence suggests that the partitioning system couples plasmid segregation to chromosome segregation during mitosis. However, the coupling mechanism has not been elucidated. In order to probe into this question more incisively, we have characterized the segregation of a single-copy STB reporter plasmid by manipulating mitosis to force sister chromatids to co-segregate either without mother-daughter bias or with a finite daughter bias. We find that the STB plasmid sisters are tightly correlated to sister chromatids in the extents of co-segregation as well as the bias in co-segregation under these conditions. Furthermore, this correlation is abolished by delaying spindle organization or preventing cohesin assembly during a cell cycle. Normal segregation of the 2-micron plasmid has been shown to require spindle integrity and the cohesin complex. Our results are accommodated by a model in which spindle- and cohesin-dependent association of plasmid sisters with sister chromatids promotes their segregation by a hitchhiking mechanism.

Real-time single-molecule tethered particle motion analysis reveals mechanistic similarities and contrasts of Flp site-specific recombinase with Cre and λ Int.

Flp, a tyrosine site-specific recombinase coded for by the selfish two micron plasmid of Saccharomyces cerevisiae, plays a central role in the maintenance of plasmid copy number. The Flp recombination system can be manipulated to bring about a variety of targeted DNA rearrangements in its native host and under non-native biological contexts. We have performed an exhaustive analysis of the Flp recombination pathway from start to finish by using single-molecule tethered particle motion (TPM). The recombination reaction is characterized by its early commitment and high efficiency, with only minor detraction from 'non-productive' and 'wayward' complexes. The recombination synapse is stabilized by strand cleavage, presumably by promoting the establishment of functional interfaces between adjacent Flp monomers. Formation of the Holliday junction intermediate poses a rate-limiting barrier to the overall reaction. Isomerization of the junction to the conformation favoring its resolution in the recombinant mode is not a slow step. Consistent with the completion of nearly every initiated reaction, the chemical steps of strand cleavage and exchange are not reversible during a recombination event. Our findings demonstrate similarities and differences between Flp and the mechanistically related recombinases λ Int and Cre. The commitment and directionality of Flp recombination revealed by TPM is consistent with the physiological role of Flp in amplifying plasmid DNA.

Temporal sequence and cell cycle cues in the assembly of host factors at the yeast 2 micron plasmid partitioning locus.

The Saccharomyces cerevisiae 2 micron plasmid exemplifies a benign but selfish genome, whose stability approaches that of the chromosomes of its host. The plasmid partitioning locus STB (stability locus) displays certain functional analogies with centromeres along with critical distinctions, a significant one being the absence of the kinetochore complex at STB. The remodels the structure of chromatin (RSC) chromatin remodeling complex, the nuclear motor Kip1, the histone H3 variant Cse4 and the cohesin complex associate with both loci. These factors appear to contribute to plasmid segregation either directly or indirectly through their roles in chromosome segregation. Assembly and disassembly of the plasmid-coded partitioning proteins Rep1 and Rep2 and host factors at STB follow a temporal hierarchy during the cell cycle. Assembly is initiated by STB association of [Rsc8-Rsc58], followed by [Rep1-Rep2-Kip1] and [Cse4-Rsc2-Sth1] recruitment, and culminates in cohesin assembly. Disassembly starts with dissociation of RSC components, is followed by cohesin disassembly and Cse4 exit during anaphase and late telophase, respectively. [Rep1-Rep2-Kip1] persists through G1 of the ensuing cell cycle. The de novo assembly of the 'partitioning complex' is cued by the innate cell cycle clock and is dependent on DNA replication. Shared functional attributes of STB and centromere (CEN) are consistent with a potential evolutionary link between them.

Histone H3-variant Cse4-induced positive DNA supercoiling in the yeast plasmid has implications for a plasmid origin of a chromosome centromere.

The Saccharomyces cerevisiae 2-µm plasmid is a multicopy selfish genome that resides in the nucleus. The genetic organization of the plasmid is optimized for stable, high-copy propagation in host-cell populations. The plasmid's partitioning system poaches host factors, including the centromere-specific histone H3-variant Cse4 and the cohesin complex, enabling replicated plasmid copies to segregate equally in a chromosome-coupled fashion. We have characterized the in vivo chromatin topology of the plasmid partitioning locus STB in its Cse4-associated and Cse4-nonassociated states. We find that the occupancy of Cse4 at STB induces positive DNA supercoiling, with a linking difference (ΔLk) contribution estimated between +1 and +2 units. One plausible explanation for this contrary topology is the presence of a specialized Cse4-containing nucleosome with a right-handed DNA writhe at a functional STB, contrasted by a standard histone H3-containing nucleosome with a left-handed DNA writhe at a nonfunctional STB. The similarities between STB and centromere in their nucleosome signature and DNA topology would be consistent with the potential origin of the unusual point centromere of budding yeast chromosomes from the partitioning locus of an ancestral plasmid.

Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination.

Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site-specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3'-phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg-308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti-hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site-specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns.

Topological similarity between the 2µm plasmid partitioning locus and the budding yeast centromere: evidence for a common evolutionary origin?

The partitioning locus STB of the selfish plasmid, the 2µm circle, of Saccharomyces cerevisiae is essential for the propagation of this multi-copy extra-chromosomal DNA element with nearly chromosome-like stability. The functional competence of STB requires the plasmid-coded partitioning proteins Rep1 and Rep2 as well as host-coded proteins. Host factors that associate with STB in a Rep1- and Rep2-dependent manner also interact with centromeres, and play important roles in chromosome segregation. They include the cohesin complex and the centromere-specific histone H3 variant Cse4. The genetically defined point centromere of S. cerevisiae differs starkly from the much more widespread epigenetically specified regional centromeres of eukaryotes. The particularly small size of the S. cerevisiae centromere and the association of chromosome segregation factors with STB raise the possibility of an evolutionary link between these two partitioning loci. The unusual positive supercoiling harboured by the S. cerevisiae centromere and STB in vivo in their functional states, unveiled by recent experiments, bolsters the notion of their potential descent from an ancestral plasmid partitioning locus.

The 2 micron plasmid of Saccharomyces cerevisiae: a miniaturized selfish genome with optimized functional competence.

The 2 micron plasmid of Saccharomyces cerevisiae is a relatively small multi-copy selfish DNA element that resides in the yeast nucleus at a copy number of 40-60 per haploid cell. The plasmid is able to persist in host populations with almost chromosome-like stability with the help of a partitioning system and a copy number control system. The first part of this article describes the properties of the partitioning system comprising two plasmid coded proteins, Rep1 and Rep2, and a partitioning locus STB. Current evidence supports a model in which the Rep-STB system couples plasmid segregation to chromosome segregation by promoting the physical association of plasmid molecules with chromosomes. In the second part, the focus is on the Flp site-specific recombination system housed by the plasmid, which plays a critical role in maintaining steady state plasmid copy number. The Flp system corrects any decrease in plasmid population by promoting plasmid amplification via a recombination induced rolling circle replication mechanism. Appropriate plasmid amplification, without runaway increase in copy number, is ensured by positive and negative regulation of FLP gene expression by plasmid coded proteins and by the control of Flp level/activity through post-translational modification of Flp by the cellular sumoylation system. The Flp system has been successfully utilized to understand mechanisms of site-specific recombination and to bring about directed genetic alterations for addressing fundamental problems in biology and for accomplishing bio-engineering objectives. A particularly interesting, and perhaps less well known and underappreciated, application of Flp in revealing unique DNA topologies required to confer functional competence to DNA-protein machines is discussed.

Yeast cohesin complex embraces 2 micron plasmid sisters in a tri-linked catenane complex.

Sister chromatid cohesion, crucial for faithful segregation of replicated chromosomes in eukaryotes, is mediated by the multi-subunit protein complex cohesin. The Saccharomyces cerevisiae plasmid 2 micron circle mimics chromosomes in assembling cohesin at its partitioning locus. The plasmid is a multi-copy selfish DNA element that resides in the nucleus and propagates itself stably, presumably with assistance from cohesin. In metaphase cell lysates, or fractions enriched for their cohesed state by sedimentation, plasmid molecules are trapped topologically by the protein ring formed by cohesin. They can be released from cohesin's embrace either by linearizing the DNA or by cleaving a cohesin subunit. Assays using two distinctly tagged cohesin molecules argue against the hand-cuff (an associated pair of monomeric cohesin rings) or the bracelet (a dimeric cohesin ring) model as responsible for establishing plasmid cohesion. Our cumulative results most easily fit a model in which a single monomeric cohesin ring, rather than a series of such rings, conjoins a pair of sister plasmids. These features of plasmid cohesion account for its sister-to-sister mode of segregation by cohesin disassembly during anaphase. The mechanistic similarities of cohesion between mini-chromosome sisters and 2 micron plasmid sisters suggest a potential kinship between the plasmid partitioning locus and centromeres.

Restoration of catalytic functions in Cre recombinase mutants by electrostatic compensation between active site and DNA substrate.

Two conserved catalytic arginines, Arg-173 and Arg-292, of the tyrosine site-specific recombinase Cre are essential for the transesterification steps of strand cleavage and joining in native DNA substrates containing scissile phosphate groups. The active site tyrosine (Tyr-324) provides the nucleophile for the cleavage reaction, and forms a covalent 3'-phosphotyrosyl intermediate. The 5'-hydroxyl group formed during cleavage provides the nucleophile for the joining reaction between DNA partners, yielding strand exchange. Previous work showed that substitution of the scissile phosphate (P) by methylphosphonate (MeP) permits strand cleavage by a Cre variant lacking Arg-292. We now demonstrate that MeP activation and cleavage are not blocked by substitution of Arg-173 or even simultaneous substitutions of Arg-173 and Arg-292 by alanine. Furthermore, Cre(R173A) and Cre(R292A) are competent in strand joining, Cre(R173A) being less efficient. No joining activity is detected with Cre(R173A, R292A). Consistent with their ability to cleave and join strands, Cre(R173A) and Cre(R292A) can promote recombination between two MeP-full-site DNA partners. These findings shed light on the overall contribution of active site electrostatics, and tease apart distinctive contributions of the individual arginines, to the chemical steps of recombination. They have general implications in active site mechanisms that promote important phosphoryl transfer reactions in nucleic acids.

Electrostatic suppression allows tyrosine site-specific recombination in the absence of a conserved catalytic arginine.

The active site of the tyrosine family site-specific recombinase Flp contains a conserved catalytic pentad that includes two arginine residues, Arg-191 and Arg-308. Both arginines are essential for the transesterification steps of strand cleavage and strand joining in DNA substrates containing a phosphate group at the scissile position. During strand cleavage, the active site tyrosine supplies the nucleophile to form a covalent 3'-phosphotyrosyl intermediate. The 5'-hydroxyl group produced by cleavage provides the nucleophile to re-form a 3'-5' phosphodiester bond in a recombinant DNA strand. In previous work we showed that substitution of the scissile phosphate (P) by the charge neutral methylphosphonate (MeP) makes Arg-308 dispensable during the catalytic activation of the MeP diester bond. However, in the Flp(R308A) reaction, water out-competes the tyrosine nucleophile (Tyr-343) to cause direct hydrolysis of the MeP diester bond. We now report that for MeP activation Arg-191 is also not required. In contrast to Flp(R308A), Flp(R191A) primarily mediates normal cleavage by Tyr-343 but also exhibits a weaker direct hydrolytic activity. The cleaved MeP-tyrosyl intermediate formed by Flp(R191A) can be targeted for nucleophilic attack by a 5'-hydroxyl or water and channeled toward strand joining or hydrolysis, respectively. In collaboration with wild type Flp, Flp(R191A) promotes strand exchange between MeP- and P-DNA partners. Loss of a catalytically crucial positively charged side chain can thus be suppressed by a compensatory modification in the DNA substrate that neutralizes the negative charge on the scissile phosphate.