Predicting knot or catenane type of site-specific recombination products

Site-specific recombination on supercoiled circular DNA yields a variety of knotted or catenated products. Here, we present a topological model of this process and characterize all possible products of the most common substrates: unknots, unlinks, and torus knots and catenanes. This model tightly prescribes the knot or catenane type of previously uncharacterized data. We also discuss how the model helps to distinguish products of distributive recombination and, in some cases, determine the order of processive recombination products.     

Unveiling hidden catalytic contributions of the conserved His/Trp-III in tyrosine recombinases: assembly of a novel active site in Flp recombinase harboring alanine at this position

The catalytic pentad of tyrosine recombinases, that assists the tyrosine nucleophile, includes a conserved histidine/tryptophan (His/Trp-III). Flp and Cre harbor tryptophan at this position; most of their kin recombinases display histidine. Contrary to the conservation rule, Flp(W330F) is a much stronger recombinase than Flp(W330H). The hydrophobicity of Trp330 or Phe330 is utilized in correctly positioning Tyr343 during the strand cleavage step of recombination. Why then is phenylalanine almost never encountered in the recombinase family at this conserved position? Using exogenous nucleophiles and synthetic methylphosphonate or 5'-thiolate substrates, we decipher that Trp330 also assists in the activation of the scissile phosphate and the departure of the 5'-hydroxyl leaving group. These two functions are consistent with the hydrogen bonding property of Trp330 as well as its location in structures of the Flp recombination complexes. However, van der Waals contact between Trp330 and Arg308 may also be important for the phosphate activation step. A structure based suppression strategy permits the inactive variant Flp(W330A) to be rescued by a second site mutation A339M. Modeling alanine and methionine at positions 330 and 339, respectively, in the Flp crystal structure suggests a plausible mechanism for active site restoration. Successful suppression suggests the possibility of evolving, by design, new active site configurations for tyrosine recombination.     

DNA topology and geometry in Flp and Cre recombination

The Flp recombinase of yeast and the Cre recombinase of bacteriophage P1 both belong to the lambda-integrase (Int) family of site-specific recombinases. These recombination systems recognize recombination-target sequences that consist of two 13bp inverted repeats flanking a 6 or 8bp spacer sequence. Recombination reactions involve particular geometric and topological relationships between DNA target sites at synapsis, which we investigate using nicked-circular DNA molecules. Examination of the tertiary structure of synaptic complexes formed on nicked plasmid DNAs by atomic-force microscopy, in conjunction with detailed topological analysis using the mathematics of tangles, shows that only a limited number of recombination-site topologies are consistent with the global structures of plasmids bearing directly and inversely repeated sites. The tangle solutions imply that there is significant distortion of the Holliday-junction intermediate relative to the planar structure of the four-way DNA junction present in the Flp and Cre co-crystal structures. Based on simulations of nucleoprotein structures that connect the two-dimensional tangle solutions with three-dimensional models of the complexes, we propose a recombination mechanism in which the synaptic intermediate is characterized by a non-planar, possibly near-tetrahedral, Holliday-junction intermediate. Only modest conformational changes within this structure are needed to form the symmetric, planar DNA junction, which may be characteristic of shorter-lived intermediates along the recombination pathway.     

Stepwise manipulation of DNA specificity in Flp recombinase: progressively adapting Flp to individual and combinatorial mutations in its target site

The Flp protein from Saccharomyces cerevisiae is one of the site-specific tyrosine family recombinases that are used widely in genomic engineering. As a first step towards mediating directed DNA rearrangements at non-native Flp recombination targets (mFRTs), we have evolved three separate groups of Flp variants that preferentially act on mFRTs containing substitutions at the first, seventh or both positions of the Flp-binding elements. The variants that recombine the double-mutant mFRT contain a subset of the mutations present in those that are active on the single-mutant mFRTs, plus additional mutations. Specificity for and discrimination between target sites, effected primarily by amino acid residues that contact DNA, can be modulated by those that do not interact with DNA or with a DNA-contacting residue. The degree of modulation can range from relaxed DNA specificity to almost completely altered specificity. Our results suggest that combined DNA shuffling and mutagenesis of libraries of Flp variants active on distinct mFRTs can yield variants that can recombine mFRTs containing combinations of the individual mutations.     

Determinants of product topology in a hybrid Cre-Tn3 resolvase site-specific recombination system

Many natural DNA site-specific recombination systems achieve directionality and/or selectivity by making recombinants with a specific DNA topology. This property requires that the DNA architecture of the synapse and the mechanism of strand exchange are both under strict control. Previously we reported that Tn3 resolvase-mediated synapsis of the accessory binding sites from the Tn3 recombination site res can impose topological selectivity on Cre/loxP recombination. Here, we show that the topology of these reactions is profoundly affected by subtle changes in the hybrid recombination site les. Reversing the orientation of loxP relative to the res accessory sequence, or adding 4 bp to the DNA between loxP and the accessory sequence, can switch between two-noded and four-noded catenane products. By analysing Holliday junction intermediates, we show that the innate bias in the order of strand exchanges at loxP is maintained despite the changes in topology. We conclude that a specific synaptic structure formed by resolvase and the res accessory sequences permits Cre to align the adjoining loxP sites in several distinct ways, and that resolvase-mediated intertwining of the accessory sequences may be less than has been assumed previously.     

镜像克隆系统:DNA重组技术的新进展

镜像克隆系统是近几年新发展起来的一种DNA重组技术,它突破了传统重组限制酶切及连接的繁琐和费时,利用重组酶使外源基因快速、方便地进行亚克隆和表达。本阐述了镜像克隆系统的结构、作用原理及特点。     

Sister chromatid interactions in bacteria revealed by a site-specific recombination assay

The process of Sister Chromosome Cohesion (SCC), which holds together sister chromatids upon replication, is essential for chromosome segregation and DNA repair in eukaryotic cells. Although cohesion at the molecular level has never been described in E. coli, previous studies have reported that sister sequences remain co-localized for a period after their replication. Here, we have developed a new genetic recombination assay that probes the ability of newly replicated chromosome loci to interact physically. We show that Sister Chromatid Interaction (SCI) occurs exclusively within a limited time frame after replication. Importantly, we could differentiate sister cohesion and co-localization since factors such as MatP and MukB that reduced the co-localization of markers had no effect on molecular cohesion. The frequency of sister chromatid interactions were modulated by the activity of Topo-IV, revealing that DNA topology modulates cohesion at the molecular scale in bacteria.     

Reversed DNA strand cleavage specificity in initiation of Cre-LoxP recombination induced by the His289Ala active-site substitution

During the first steps of site-specific recombination, Cre protein cleaves and religates a specific homologous pair of LoxP strands to form a Holliday junction (HJ) intermediate. The HJ is resolved into recombination products through exchange of the second homologous strand pair. CreH289A, containing a His to Ala substitution in the conserved R-H-R catalytic motif, has a 150-fold reduced recombination rate and accumulates HJs. However, to produce these HJs, CreH289A exchanges the opposite set of strands compared to wild-type Cre (CreWT). To investigate how CreH289A and CreWT impose strand exchange order, we characterized their reactivities and strand cleavage preferences toward LoxP duplex and HJ substrates containing 8bp spacer substitutions. Remarkably, CreH289A had different and often opposite strand exchange preferences compared to CreWT with nearly all substrates. CreH289N was much less perturbed, implying that overall recombination rate and strand exchange depend more on His289 hydrogen bonding capability than on its acid/base properties. LoxP substitutions immediately 5' (S1 nucleotide) or 3' (S1' nucleotide) of the scissile phosphate had large effects on substrate utilization and strand exchange order. S1' substitutions, designed to alter base-unstacking events concomitant with Cre-induced LoxP bending, caused HJ accumulation and dramatically inverted the cleavage preferences. That pre-formed HJs were resolved via either strand in vitro suggests that inhibition of the "conformational switch" isomerization required to trigger the second strand exchange accounts for the observed HJ accumulation. Rather than reflecting CreWT behavior, CreH289A accumulates HJs of opposite polarity through a combination of its unique cleavage specificity and an HJ isomerization defect. The overall implication is that cleavage specificity is mediated by sequence-dependent DNA deformations that influence the scissile phosphate positioning and reactivity. A role of His289 may be to selectively stabilize the "activated" phosphate conformation in order to promote cleavage.     

Symmetric DNA sites are functionally asymmetric within Flp and Cre site-specific DNA recombination synapses

Flp and Cre-mediated recombination on symmetrized FRT and loxP sites, respectively, in circular plasmid substrates yield both DNA inversion and deletion. However, upon sequestering three negative supercoils outside the recombination complex using the resII-resIII synapse formed by Tn3 resolvase and the LER synapse formed by phage Mu transposase in the case of Flp and Cre, respectively, the reactions are channeled towards inversion at the expense of deletion. The inversion product is a trefoil, its unique topology being conferred by the external resolvase or LER synapse. Thus, Flp and Cre assign their symmetrized substrates a strictly antiparallel orientation with respect to strand cleavage and exchange. These conclusions are supported by the product profiles from tethered parallel and antiparallel native FRT sites in dilution and competition assays. Furthermore, the observed recombination bias favoring deletion over inversion in a nicked circular substrate containing two symmetrized FRT sites is consistent with the predictions from Monte Carlo simulations based on antiparallel synapsis of the DNA partners.     

Analysis of insertion into secondary attachment sites by phage lambda and by int mutants with altered recombination specificity

When phage lambda lysogenizes a cell that lacks the primary bacterial attachment site, integrase catalyzes insertion of the phage chromosome into one of many secondary sites. Here, we characterize the secondary sites that are preferred by wild-type lambda and by lambda int mutants with altered insertion specificity. The sequences of these secondary sites resembled that of the primary site: they contained two imperfect inverted repeats flanking a short spacer. The imperfect inverted repeats of the primary site bind integrase, while the 7 bp spacer, or overlap region, swaps strands with a complementary sequence in the phage attachment site during recombination. We found substantial sequence conservation in the imperfect inverted repeats of secondary sites, and nearly perfect conservation in the leftmost three bases of the overlap region. By contrast, the rightmost bases of the overlap region were much more variable. A phage with an altered overlap region preferred to insert into secondary sites with the corresponding bases. We suggest that this difference between the left and right segments is a result of the defined order of strand exchanges during integrase-promoted recombination. This suggestion accounts for the unexpected segregation pattern of the overlap region observed after insertion into several secondary sites. Some of the altered specificity int mutants differed from wild-type in secondary site preference, but we were unable to identify simple sequence motifs that account for these differences. We propose that insertion into secondary sites is a step in the evolutionary change of phage insertion specificity and present a model of how this might occur.     

Tangle analysis of Xer recombination reveals only three solutions, all consistent with a single three-dimensional topological pathway

The product of Xer recombination at directly repeated psi sites on a circular unknotted DNA molecule is a right-hand four-noded catenane. Here, we use tangle equations to analyze the topological changes associated with Xer recombination at psi. This mathematical method allows computation of all possible topological pathways consistent with the experimental data. We give a rigorous mathematical proof that, under reasonable biological assumptions, there are only three solutions to the tangle equations. One of the solutions corresponds to a synaptic complex with antiparallel alignment of recombination core sites, the other two correspond to parallel alignment of cores. We show that all three solutions can be unified into a single three-dimensional model for Xer recombination. Thus the three distinct mathematical solutions do not necessarily represent distinct three-dimensional pathways, and in this case the three distinct tangle solutions are different planar projections of the same three-dimensional configuration.     

Site-specific recombination in eukaryotic cells mediated by mutant lambda integrases: implications for synaptic complex formation and the reactivity of episomal DNA segments

Mutant lambda integrases catalyze site-specific DNA recombination in the absence of accessory factors IHF, XIS, and negative DNA supercoiling. Here we investigate the effects that a human cellular environment exerts on these reactions in order to (i) gain further insights into mechanistic aspects of recombination in eukaryotic cells and (ii) to further develop the Int system for biotechnological applications. First, we compared intra- and intermolecular integrative as well as excisive recombination pathways on episomal substrates after co-transfection with recombinase expression vectors. Our results demonstrate that, within 24 hours after transfection, intermolecular recombination by mutant integrase is at least as efficient as intramolecular recombination. Second, a significant intermolecular recombination activity was observed between two copies of a recombination site containing only the 21 bp comprising core-type DNA sequence. This basic activity was stimulated several-fold when arm-type DNA sequences were present in addition to core sites. Therefore, one recombination pathway in human cells involves mutant integrases bound solely at core sites, which is reminiscent of the Flp/FRT and Cre/loxP pathways. The stimulatory effect of arm-type sequences could be explained by an increase in integrase concentration in the vicinity of core sites. We show, in addition, that an N-terminal truncated mutant integrase exhibited only a very weak recombinogenic activity in a eukaryotic background. This result strengthens a functional role for the N-terminal domain in recombination in addition to its arm-type DNA-binding activity. Finally, we demonstrate that low level integrative recombination by wild-type integrase is stimulated when purified integration host factor is co-transfected. This corroborates our previous conclusion that sufficient amounts of eukaryotic protein co-factors, which could functionally replace IHF, are not present in human cells. It also provides a potential means to control site-specific recombination in eukaryotic cells.     

Regulation of directionality in bacteriophage lambda site-specific recombination: structure of the Xis protein

Upon induction of a bacteriophage lambda lysogen, a site-specific recombination reaction excises the phage genome from the chromosome of its bacterial host. A critical regulator of this process is the phage-encoded excisionase (Xis) protein, which functions both as a DNA architectural factor and by cooperatively recruiting integrase to an adjacent binding site specifically required for excision. Here we present the three-dimensional structure of Xis and the results of a structure-based mutagenesis study to define the molecular basis of its function. Xis adopts an unusual "winged"-helix motif that is modeled to interact with the major- and minor-grooves of its binding site through a single alpha-helix and loop structure ("wing"), respectively. The C-terminal tail of Xis, which is required for cooperative binding with integrase, is unstructured in the absence of DNA. We propose that asymmetric bending of DNA by Xis positions its unstructured C-terminal tail for direct contacts with the N-terminal DNA-binding domain of integrase and that an ensuing disordered to ordered transition of the tail may act to stabilize the formation of the tripartite integrase-Xis-DNA complex required for phage excision.     

Asymmetric removal of supercoils suggests how topoisomerase II simplifies DNA topology

Type-IIA topoisomerases consume ATP as they catalyse the interconversion of DNA topoisomers by transporting one DNA segment through a transient break in another. It remains unclear how their activity simplifies the topology of DNA below equilibrium values. Here we report that eukaryotic topoisomerase II narrows the thermal distribution of DNA supercoils, by mainly removing negative DNA crossings. Surprisingly, this asymmetry in supercoil removal is not due to deformation of the DNA before strand passage. Topoisomerase II neither bends nor alters the helical conformation of the interacting DNA. Rather, it appears to interact with a third DNA segment, in addition to the gated and the transported segments. Remarkably, the simultaneous interaction with three DNA segments accounts for the asymmetric removal of supercoils in relaxed DNA and gives a clue to how topoisomerase II simplifies the topology of DNA against the thermal drive.     

Developmentally programmed excision of internal DNA sequences in Paramecium aurelia

The development of a new somatic nucleus (macronucleus) during sexual reproduction of the ciliate Paramecium aurelia involves reproducible chromosomal rearrangements that affect the entire germline genome. Macronuclear development can be induced experimentally, which makes P. aurelia an attractive model for the study of the mechanism and the regulation of DNA rearrangements. Two major types of rearrangements have been identified: the fragmentation of the germline chromosomes, followed by the formation of the new macronuclear chromosome ends in association with imprecise DNA elimination, and the precise excision of internal eliminated sequences (IESs). All IESs identified so far are short, A/T rich and non-coding elements. They are flanked by a direct repeat of a 5’-TA-3’ dinucleotide, a single copy of which remains at the macronuclear junction after excision. The number of these single-copy sequences has been estimated to be around 60 000 per haploid genome. This review focuses on the current knowledge about the genetic and epigenetic determinants of IES elimination in P. aurelia, the analysis of excision products, and the tightly regulated timing of excision throughout macronuclear development. Several models for the molecular mechanism of IES excision will be discussed in relation to those proposed for DNA elimination in other ciliates.     

Interactions of protein complexes on supercoiled DNA: the mechanism of selective synapsis by Tn3 resolvase

"Looping" interactions of distant sites on DNA molecules, mediated by DNA-binding proteins, feature in many regulated genetic processes. We used plasmids containing up to six res recombination sites for Tn3 resolvase to analyse looping interactions (synapsis) in this system. We observed that in plasmids with four or more res sites, certain pairs of sites recombine faster than others. The relative rates of recombination depend on the number, relative orientation, and arrangement of the sites. To account for the differences in rate, we propose that pairing interactions between resolvase-bound res sites are in a state of rapid flux, leading to configurations in which the maximum number of sites within each supercoiled substrate molecule are synapsed in a topologically simple arrangement. Recombination rates reflect the steady state concentrations of these synapse configurations. Our results are at variance with models for selective synapsis that rely on ordered motions within supercoiled DNA, "slithering" or "tracking", but are compatible with models that call for reversible synapsis of pairs of sites by random collision, followed by formation of an interwound productive synapse.     

Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences

Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There were no indications that the tomato DNA inserts conferred autonomous replication on the plasmids. Instead, Southern blot hybridization analysis of seven kanamycin resistant calli revealed the presence of at least one kanamycin resistance locus per transformant integrated in the tomato nuclear DNA. Generally one to three truncated plasmid copies were found integrated into the tomato nuclear DNA, often physically linked to each other. For one transformant we have been able to use the bacterial ampicillin resistance marker of the vector plasmid pUC9 to rescue a recombinant plasmid from the tomato genome. Analysis of the foreign sequences included in the rescued plasmid showed that integration had occurred in a non-repetitive DNA region. Calf-thymus DNA, used as a carrier in transformation procedure, was found to be covalently linked to plasmid DNA sequences in the genomic DNA of one transformant. A model is presented describing the fate of exogenously added DNA during the transformation of a plant cell. The results are discussed in reference to the possibility of isolating DNA sequences responsible for autonomous replication in tomato.     

Reversible dimer formation and stability of the anti-tumour single-chain Fv antibody MFE-23 by neutron scattering,analytical ultracentrifugation,and NMR and FT-IR spectroscopy

Cre recombinase uses two pairs of sequential cleavage and religation reactions to exchange homologous DNA strands between 34 base-pair (bp) LoxP recognition sequences. In the oligomeric recombination complex, a switch between "cleaving" and "non-cleaving" subunit conformations regulates the number, order, and regio-specificity of the strand exchanges. However, the particular sequence of events has been in question. From analysis of strand composition of the Holliday junction (HJ) intermediate, we determined that Cre initiates recombination of LoxP by cleaving the upper strand on the left arm. Cre preferred to react with the left arm of a LoxP suicide substrate, but at a similar rate to the right arm, indicating that the first strand to be exchanged is selected prior to cleavage. We propose that during complex assembly the cleaving subunit preferentially associates with the LoxP left arm, directing the first strand exchange to that side. In addition, this biased assembly would enforce productive orientation of LoxP sites in the recombination synapses. A novel Cre-HJ complex structure in which LoxP was oriented with the left arm bound by the cleaving Cre subunit suggested a physical basis for the strand exchange order. Lys86 and Lys201 interact with the left arm scissile adenine base differently than in structures that have a scissile guanine. These interactions are associated with positioning the 198-208 loop, a structural component of the conformational switch, in a configuration that is specific to the cleaving conformation. Our results suggest that strand exchange order and site alignment are regulated by an "induced fit" mechanism in which the cleaving conformation is selectively stabilized through protein-DNA interactions with the scissile base on the strand that is cleaved first.