Asymmetry in Flp-mediated cleavage

Flp is a member of the integrase family of site-specific recombinases. Members of the integrase family mediate DNA strand cleavage via a transesterification reaction involving an active site tyrosine residue. The first step of the reaction results in covalent linkage of the protein to the 3'-phosphoryl DNA terminus, leaving a 5'-hydroxyl group at the site of the nick. We have used Flp recognition target (FRT) sites containing a 5'-bridging phosphorothioate linkage at the site of Flp cleavage to accumulate intermediates in which Flp is covalently bound at a cleavage site. We have probed these intermediates with dimethylsulfate using methylation protection and find that Flp-mediated cleavage is associated with protection of two adenine residues that are opposite the sites of cleavage and covalent attachment by Flp. Methylation interference studies showed that cleavage and covalent attachment are also accompanied by differences in the contacts of Flp with each of the two cleavage sites and with the surrounding symmetry elements. Therefore, we provide evidence that Flp-mediated cleavage and covalent attachment result in changes to the conformation of the Flp-FRT complex. These changes may be required for Flp-mediated strand exchange activity.     

The Flp recombinase cleaves Holliday junctions in trans

The Flp site-specific recombinase is encoded by the 2 micrometers plasmid Saccharomyces cerevisiae and is a member of the integrase family of recombinases. Like all members of the integrase family studied, Flp mediates recombination in two steps. First, a pair of strand exchanges creates a Holliday-like intermediate; second, this intermediate is resolved to recombinant products by a second pair of strand exchanges. Evidence derived from experiments using linear substrates indicates that Flp's active site is composed of two Flp protomers. One binds to the Flp recognition target site (FRT site) and activates the scissile phosphodiester bond for cleavage. Another molecule of Flp bound elsewhere in the synaptic complex (in trans) donates the nucleophilic tyrosine that executes cleavage and thereby becomes covalently attached to the 3' phosphoryl group at the cleavage site. It has previously been shown that Flp efficiently resolves synthetic, Holliday-like (chi) structures to linear products. In this paper, we examined whether resolution of chi structures by Flp also occurs via the trans cleavage mechanism. We used in vitro complementation studies of mutant Flp proteins as well as nicked chi structures to show that Flp resolves chi structures by trans cleavage. We propose a model for Flp-mediated recombination that incorporates trans cleavage at both the initial and resolution steps of strand exchange.     

Role of partner homology in DNA recombination. Complementary base pairing orients the 5'-hydroxyl for strand joining during Flp site-specific recombination

Absolute homology between partner substrates within the strand exchange region is an essential requirement for recombination mediated by the yeast site-specific recombinase Flp. Using combinations of specially designed half- and full-site Flp substrates, we demonstrate that the strand joining step of recombination is exquisitely sensitive to spacer homology. At each exchange point, 2-3 spacer nucleotides adjacent to the nick within the cleaved strand of one substrate must base pair with the corresponding segment of the un-nicked strand from the second substrate for efficient strand joining in the recombinant mode. In accordance with the "cis-activation/trans-nucleophilic attack" model for each of the two transesterification steps of Flp recombination (strand cleavage and strand joining), we propose that the limited strand pairing orients the DNA-nucleophile (5'-hydroxyl) for attack on its target diester (3'-phosphotyrosyl-Flp). During one round of recombination, 4-6 terminal base pairs of the spacer (2-3 base pairs at each spacer end) must unpair, following strand cleavage, within a DNA substrate and pair with the partner substrate prior to strand union. In this model, the extent of branch migration of the covalently closed Holliday intermediate is limited to the central core of the spacer. The templated positioning of reactive nucleic acid groups (which is central to the model) may be utilized by other recombination systems and by RNA splicing reactions.     

Circularization and cleavage of guinea pig cytomegalovirus genomes

The mechanisms by which herpesvirus genome ends are fused to form circles after infection and are re-formed by cleavage from concatemeric DNA are unknown. We used the simple structure of guinea pig cytomegalovirus genomes, which have either one repeated DNA sequence at each end or one repeat at one end and no repeat at the other, to study these mechanisms. In circular DNA, two restriction fragments contained fused terminal sequences and had sizes consistent with the presence of single or double terminal repeats. This result implies a simple ligation of genomic ends and shows that circularization does not occur by annealing of single-stranded terminal repeats formed by exonuclease digestion. Cleavage to form the two genome types occurred at two sites, and homologies between these sites identified two potential cis elements that may be necessary for cleavage. One element coincided with the A-rich region of a pac2 sequence and had 9 of 11 bases identical between the two sites. The second element had six bases identical at both sites, in each case 7 bp from the termini. To confirm the presence of cis cleavage elements, a recombinant virus in which foreign sequences displaced the 6- and 11-bp elements 1 kb from the cleavage point was constructed. Cleavage at the disrupted site did not occur. In a second recombinant virus, restoration of 64 bases containing the 6- and 11-bp elements to the disrupted cleavage site restored cleavage. Therefore, cis cleavage elements exist within this 64-base region, and sequence conservation suggests that they are the 6- and 11-bp elements.     

Determinants of the position of a Flp-induced DNA bend

The Flp site-specific recombinase from Saccharomyces cerevisiae induces DNA bending upon interaction with the Flp recognition target (FRT) site. The minimal FRT site is comprised of two inverted binding elements which flank a central core region. Binding of a single monomer of Flp to DNA induces a DNA bend of 60 degrees. The position of this bend differed depending on whether the substrate contained a single binding element or a two-element FRT site. In the present work we tested and disproved a model in which a single Flp monomer interacts with both symmetry elements of a single FRT site. Likewise, we showed that a model in which a Flp monomer dissociates from a singly occupied FRT site and reassociates with the unbound element of another singly occupied FRT site during electrophoresis, does not account for the apparent shift in the position of the bend centre. It seems that the movement of a Flp monomer between the a and b elements of one FRT site during electrophoresis accounts for this anomaly. The position of the DNA bend resulting from the association of a Flp monomer with the FRT site is also influenced by the DNA sequences flanking the site. We conclude that attempts to measure the bend centre of a complex of one Flp molecule bound to a DNA containing two binding elements give misleading results. The position of the bend is more accurately measured in the presence of a single binding element.     

Analysis of a core domain in Drosophila DNA topoisomerase II. Targeting of an antitumor agent ICRF-159

To investigate the biochemical properties of individual domains of eukaryotic topoisomerase (topo) II, two truncation mutants of Drosophila topo II were generated, ND406 and core domain. Both mutants lack the ATPase domain, corresponding to the N-terminal 406 amino acid residues in Drosophila protein. The core domain also lacks 240 amino acid residues of the hydrophilic C-terminal region. The mutant proteins have lost DNA strand passage activity while retaining the ability to cleave the DNA and the sequence preference in protein/DNA interaction. The cleavage experiments carried out in the presence of several topo II poisons suggest that the core domain is the key target for these drugs. We have used glass-fiber filter binding assay and CsCl density gradient ultracentrifugation to monitor the formation of a salt-stable, protein-clamp complex. Both truncation mutant proteins can form a clamp complex in the presence of an antitumor agent, ICRF-159, suggesting that the drug targets the core domain of the enzyme and promotes the intradimeric closure at the N-terminal interface of the core domain. Furthermore, the salt stability of the closed protein clamp induced by ICRF-159 depends on the presence and closure of the N-terminal ATPase domain.     

Wild-type Flp recombinase cleaves DNA in trans

Site-specific recombinases of the Integrase family utilize a common chemical mechanism to break DNA strands during recombination. A conserved Arg-His-Arg triad activates the scissile phosphodiester bond, and an active-site tyrosine provides the nucleophile to effect DNA cleavage. Is the tyrosine residue for the cleavage event derived from the same recombinase monomer which provides the RHR triad (DNA cleavage in cis), or are the triad and tyrosine derived from two separate monomers (cleavage in trans)? Do all members of the family follow the same cleavage rule, cis or trans? Solution studies and available structural data have provided conflicting answers. Experimental results with the Flp recombinase which strongly support trans cleavage have been derived either by pairing two catalytic mutants of Flp or by pairing wild-type Flp and a catalytic mutant. The inclusion of the mutant has raised new concerns, especially because of the apparent contradictions in their cleavage modes posed by other Int family members. Here we test the cleavage mode of Flp using an experimental design which excludes the use of the mutant protein, and show that the outcome is still only trans DNA cleavage.     

The same two monomers within a MuA tetramer provide the DDE domains for the strand cleavage and strand transfer steps of transposition

The chemistry of Mu transposition is executed within a tetrameric form of the Mu transposase (MuA protein). A triad of DDE (Asp, Asp35Glu motif) residues in the central domain of MuA (DDE domain) is essential for both the strand cleavage and strand transfer steps of transposition. Previous studies had suggested that complete Mu transposition requires all four subunits in the MuA tetramer to carry an active DDE domain. Using a mixture of MuA proteins with either wild-type or altered att-DNA binding specificities, we have now designed specific arrangements of MuA subunits carrying the DDE domain. From analysis of the abilities of oriented tetramers to carry out DNA cleavage and strand transfer from supercoiled DNA, a new picture of the disposition of DNA and protein partners during transposition has emerged. For DNA cleavage, two subunits of MuA located at attL1 and attR1 (sites that undergo cleavage) provide DDE residues in trans. The same two subunits contribute DDE residues for strand transfer, also in trans. Thus, only two active DDE+ monomers within the tetramer carry out complete Mu transposition. We also show that when the attR1-R2 arrangement used on supercoiled substrates is tested for cleavage on linear substrates, alternative chemically competent DNA-protein associations are produced, wherein the functional DDE subunits are positioned at R2 rather than at R1.     

Cooperative binding properties of restriction endonuclease EcoRII with DNA recognition sites

EcoRII is a member of the expanding group of type IIe restriction endonucleases that share the distinguishing feature of requiring cooperativity between two recognition sites in their substrate DNA. To determine the stoichiometry of the active DNA-enzyme complex and the mode of cooperative interaction, we have investigated the dependence of EcoRII cleavage on the concentration of EcoRII dimers. Maximal restriction was observed at dimer/site ratios of 0.25 and 0. 5. The molecular weight of the DNA-enzyme complex eluted from a gel filtration column also corresponds to a dimeric enzyme structure bound to two substrate sites. We conclude that one EcoRII dimer is sufficient to interact cooperatively with two DNA recognition sites. A Lac repressor "barrier" bound between two normally reactive EcoRII sites did not inhibit restriction endonuclease activity, indicating that cooperativity between EcoRII sites is achieved by bending or looping of the intervening DNA stretch. Comparative cleavage of linear substrates with differently spaced interacting sites revealed an inverse correlation between cleavage rate and site distance. At the optimal distance of one helical turn, EcoRII cleavage is independent of the orientation of the recognition sequence in the DNA double strand.     

A general model for site-specific recombination by the integrase family recombinases

We present here a general model for integrase family site-specific recombination using the geometric relationships of the cleavable phosphodiester bonds and the disposition of the recombinase monomers (defined by their binding planes) with respect to them. The 'oscillation model' is based largely on the conformations of the recombinase-bound DNA duplexes and their dynamics within Holliday junctions. The duplex substrate or the Holliday junction intermediate is capable of 'oscillating' between two cleavage-competent asymmetric states with respect to corres-ponding chemically inert 'equilibrium positions'. The model accommodates several features of the Flp system and predicts two modes of DNA cleavage during a normal recombination event. It is equally applicable to other systems that mediate recombination across 6, 7 or 8 bp long strand exchange regions (or spacers). The model is consistent with approximately 0-1, 1-2 and 2-3 bp of branch migration during recombination reactions involving 6, 7 and 8 bp spacers, respectively.     

The catalytic domain of lambda site-specific recombinase

The Escherichia coli phage lambda integrase protein (Int) belongs to the large Int family of site-specific recombinases. It is a heterobivalent DNA binding protein that makes use of a high energy covalent phosphotyrosine intermediate to catalyze integrative and excisive recombination at specific chromosomal sites (att sites). A 293-amino acid carboxy-terminal fragment of Int (C65) has been cloned, characterized, and used to further dissect the protein. From this we have cloned and characterized a 188-amino acid, protease-resistant, carboxy-terminal fragment (C170) that we believe is the minimal catalytically competent domain of Int. C170 has topoisomerase activity and converts att suicide substrates to the covalent phosphotyrosine complexes characteristic of recombination intermediates. However, it does not show efficient binding to att site DNA in a native gel shift assay. We propose that lambda Int consists of three functional and structural domains: residues 1-64 specify recognition of "arm-type" DNA sequences distant from the region of strand exchange; residues 65-169 contribute to specific recognition of "core-type" sequences at the sites of strand exchange and possibly to protein-protein interactions; and residues 170-356 carry out the chemistry of DNA cleavage and ligation. The finding that the active site nucleophile Tyr-342 is in a uniquely protease-sensitive region complements and reinforces the recently solved C170 crystal structure, which places Tyr-342 at the center of a 17-amino acid flexible loop. It is proposed that C170 is likely to represent a generic Int family domain that thus affords a specific route to studying the chemistry of DNA cleavage and ligation in these recombinases.     

Structural alterations and conformational dynamics in Holliday junctions induced by binding of a site-specific recombinase

Binding of a cleavage-incompetent mutant of the Flp recombinase induces a roughly square-planar geometry in synthetic immobile Holliday junctions. The branch points, which are rigidly fixed in these junctions in their free forms, tend to be more flexible in their protein-bound forms. Our results (1) suggest a plausible mechanism for the switching of the recombination complex from the Holliday-forming mode to the Holliday-resolving mode, (2) provide a rationale for previous observations that Flp resolves preformed immobile Holliday structures in the parental or in the recombinant mode in a relatively unbiased manner, and (3) accommodate two modes of DNA cleavage by Flp (transhorizontal or transdiagonal) in Holliday substrates.     

The structure-selectivity and sequence-preference of the junction-resolving enzyme CCE1 of Saccharomyces cerevisiae

CCE1 is a DNA junction-resolving enzyme involved in the resolution of recombining mitochondrial DNA in Saccharomyces cerevisiae. The CCE1 gene was cloned by PCR, and the expressed protein purified to homogeneity. CCE1 was found to bind to four-way DNA junctions, with a strong structural selectivity. The enzyme binds DNA junctions as a dimer, with slow subunit exchange occurring in free solution. While CCE1 binds equally to synthetic four-way DNA junctions of any sequence, it exhibits pronounced sequence-selectivity in cleavage. Both fixed junctions and those capable of branch migration can be cleaved, with a preference for cleavage at the sequence 5'CT/. Cleavage of junctions tethered to adopt specific stacking isomers demonstrated that the target sequences are cleaved fivefold faster when located on a continuous strand compared to an exchanging strand.     

Conserved sequence and structural motifs contribute to the DNA binding and cleavage activities of a geminivirus replication protein

Tomato golden mosaic virus (TGMV), a member of the geminivirus family, has a single-stranded DNA genome that replicates through a rolling circle mechanism in nuclei of infected plant cells. TGMV encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its host. AL1 is a multifunctional protein that binds double-stranded DNA, catalyzes cleavage and ligation of single-stranded DNA, and forms oligomers. Earlier experiments showed that the region of TGMV AL1 necessary for DNA binding maps to the N-terminal 181 amino acids of the protein and overlaps the DNA cleavage (amino acids 1-120) and oligomerization (amino acids 134-181) domains. In this study, we generated a series of site-directed mutations in conserved sequence and structural motifs in the overlapping DNA binding and cleavage domains and analyzed their impact on AL1 function in vivo and in vitro. Only two of the fifteen mutant proteins were capable of supporting viral DNA synthesis in tobacco protoplasts. In vitro experiments demonstrated that a pair of predicted alpha-helices with highly conserved charged residues are essential for DNA binding and cleavage. Three sequence motifs conserved among geminivirus AL1 proteins and initiator proteins from other rolling circle systems are also required for both activities. We used truncated AL1 proteins fused to a heterologous dimerization domain to show that the DNA binding domain is located between amino acids 1 and 130 and that binding is dependent on protein dimerization. In contrast, AL1 monomers were sufficient for DNA cleavage and ligation. Together, these results established that the conserved motifs in the AL1 N terminus contribute to DNA binding and cleavage with both activities displaying nearly identical amino acid requirements. However, DNA binding was readily distinguished from cleavage and ligation by its dependence on AL1/AL1 interactions.     

The beta protein of phage lambda promotes strand exchange

Bacteriophage lambda encodes a 28 kDa protein called beta that binds to single-stranded DNA and promotes the renaturation of complementary single strands. beta Protein fails to bind directly to duplex DNA but remains bound to the DNA product of renaturation that beta itself catalyzes. These observations led to an examination of the ability of beta protein to promote strand exchange. beta Protein caused the replacement of a 43-mer oligonucleotide annealed to M13 circular single-stranded DNA by a homologous 63-mer whose 20 extra nucleotide residues were complementary to the adjacent 3' region of M13 DNA. The role of beta protein in this reaction was manifested in several ways: beta protein pushed the exchange through four to eight mismatches, which blocked exchange mediated by spontaneous renaturation and branch migration; beta imposed a polarity on the strand exchange that was lacking in the spontaneous reaction; and beta remained bound to the heteroduplex product of strand exchange. These observations reveal a mechanism by which a protein can drive strand exchange in one direction without using ATP or any other exogenous source of energy.     

Three mechanistic steps detected by FRET after presynaptic filament formation in homologous recombination. ATP hydrolysis required for release of oligonucleotide heteroduplex product from RecA

The Escherichia coli RecA protein promotes DNA strand exchange in homologous recombination and recombinational DNA repair. Stopped-flow kinetics and fluorescence resonance energy transfer (FRET) were used to study RecA-mediated strand exchange between a 30-bp duplex DNA and a homologous single-stranded 50mer. In our standard assay, one end of the dsDNA helix was labeled at apposing 5' and 3' ends with hexachlorofluorescein and fluorescein, respectively. Strand exchange was monitored by the increase in fluorescence emission resulting upon displacement of the fluorescein-labeled strand from the initial duplex. The potential advantages of FRET in study of strand exchange are that it noninvasively measures real-time kinetics in the previously inaccessible millisecond time regime and offers great sensitivity. The oligonucleotide substrates model short-range mechanistic effects that might occur within a localized region of the ternary complex formed between RecA and long DNA molecules during strand exchange. Reactions in the presence of ATP with 0.1 microM duplex and 0.1-1.0 microM ss50mer showed triphasic kinetics in 600 s time courses, implying the existence of three mechanistic steps subsequent to presynaptic filament formation. The observed rate constants for the intermediate phase were independent of the concentration of ss50mer and most likely characterize a unimolecular isomerization of the ternary complex. The observed rate constants for the first and third phases decreased with increasing ss50mer concentration. Kinetic experiments performed with the nonhydrolyzable analogue ATPgammaS showed overall changes in fluorescence emission identical to those observed in the presence of ATP. In addition, the observed rate constants for the two fastest reaction phases were identical in ATP or ATPgammaS. The observed rate constant for the slowest phase showed a 4-fold reduction in the presence of ATPgammaS. Results in ATPgammaS using an alternate fluorophore labeling pattern suggest a third ternary intermediate may form prior to ssDNA product release. The existence of two or three ternary intermediates in strand exchange with a 30 bp duplex suggests the possibility that the step size for base pair switching may be 10-15 bp. Products of reactions in the presence of ATP and ATPgammaS, with and without proteinase K treatment, were analyzed on native polyacrylamide gels. In reactions in which only short-range RecA-DNA interactions were important, ATP hydrolysis was not required for recycling of RecA from both oligonucleotide products. Hydrolysis or deproteinization was required for RecA to release the heteroduplex product, but not the outgoing single strand.     

Flp ribonuclease activities. Mechanistic similarities and contrasts to site-specific DNA recombination

The ribonuclease active site harbored by the Flp site-specific recombinase can act on two neighboring phosphodiester bonds to yield mechanistically distinct chain breakage reactions. One of the RNase reactions apparently proceeds via a covalent enzyme intermediate and targets the phosphodiester position involved in DNA recombination (Flp RNase I activity). The second activity (Flp RNase II) targets the phosphodiester immediately to the 3' side but appears not to involve an enzyme-linked intermediate. Flp RNase I is absolutely dependent upon Tyr-343 of Flp and is competitive with respect to the normal strand joining reaction. It can utilize the 2'-hydroxyl group from any one of the four ribonucleotides with comparable efficiencies in the cleavage reaction. On the other hand, the RNase II reaction mediated by Flp(Y343F) is specific for U and cannot utilize the 2'-hydroxyl group from ribo-A, -G, or -C under standard reaction conditions. The RNase II activity is also sensitive to the 3'-neighboring base. Although dT is functional, the activity is stimulated by U or U-2'-OMe. The Flp RNase II reaction effectively competes with the normal strand cleavage reaction mediated by Tyr-343, even though their phosphodiester targets are not the same.