PepA and ArgR do not regulate Cre recombination at the bacteriophage P1 loxP site

In the lysogenic state, bacteriophage P1 is maintained as a low copy-number circular plasmid. Site-specific recombination at loxP by the phage-encoded Cre protein keeps P1 monomeric, thus helping to ensure stable plasmid inheritance. Two Escherichia coli DNA-binding proteins, PepA and ArgR, were recently reported to be necessary for maintenance or establishment of P1 lysogeny. PepA and ArgR bind to regulatory DNA sequences upstream of the ColE1 cer recombination site to regulate site-specific recombination by the XerCD recombinases. This recombination keeps ColE1 in a monomeric state and helps to ensure stable plasmid maintenance. It has been suggested that ArgR and PepA play a similar role in P1 maintenance, regulating Cre recombination by binding to DNA sequences upstream of loxP. Here, we show that ArgR does not bind to its proposed binding site upstream of loxP, and that Cre recombination at loxP in its natural P1 context is not affected by PepA and ArgR in vitro. When sequences upstream of loxP were mutated to allow ArgR binding, PepA and ArgR still had no effect on Cre recombination. Our results demonstrate that PepA requires specific DNA sequences for binding, and that PepA and ArgR have no direct role in Cre recombination at P1 loxP.     

Direct interaction of aminopeptidase A with recombination site DNA in Xer site-specific recombination.

Xer site-specific recombination at ColE1 cer converts plasmid multimers into monomers, thus ensuring the heritable stability of ColE1. Two related recombinase proteins, XerC and XerD, catalyse the strand exchange reaction at a 30 bp recombination core site. In addition, two accessory proteins, PepA and ArgR, are required for recombination at cer. These two accessory proteins are thought to act at 180 bp of accessory sequences adjacent to the cer recombination core to ensure that recombination only occurs between directly repeated sites on the same molecule. Here, we demonstrate that PepA and ArgR interact directly with cer, forming a complex in which the accessory sequences of two cer sites are interwrapped approximately three times in a right-handed fashion. We present a model for this synaptic complex, and propose that strand exchange can only occur after the formation of this complex.     

Differential regulation of multiple overlapping promoters in flagellar class II operons in Escherichia coli

The cer –Xer dimer resolution system of plasmid ColE1 is highly selective, acting only at sites on the same molecule and in direct repeat. Recombination requires the XerCD recombinase and accessory proteins ArgR and PepA. The Escherichia coli chromosome dimer resolution site dif and the type II hybrid site use the same recombinase but are independent of ArgR and PepA and show no site selectivity. This has led to the proposal that ArgR and PepA are responsible for the imposition of constraint. We describe here the characterization of a novel class of 'conditionally constrained' multimer resolution sites whose properties support this hypothesis. In the presence of ArgR and PepA, plasmids containing conditionally constrained sites are monomeric, but in their absence, extensive multimerisation is seen. A mutant ArgR derivative (ArgR110), which is defective in cer -mediated dimer resolution, remains able to prevent plasmid multimerisation by a conditionally constrained site. This implies that the accessory factors block recombination in trans rather than ensuring rapid multimer resolution. When the distance between the ArgR and XerCD binding sites in a conditionally constrained site was altered by a non-integral number of helical turns, the site became unconstrained. Constraint was restored by the insertion of a full helical turn.     

ArgR and PepA, accessory proteins for XerCD-mediated resolution of ColE1 dimers, are also required for stable maintenance of the P1 prophage

Dimers of low copy number plasmids must be resolved to monomers to prevent interference with active partition. For the P1 prophage this is achieved by the Cre site-specific recombinase acting at lox. Multimerisation of multicopy plasmids threatens stability via copy number depression, and multimers of ColE1 are resolved by XerCD-mediated recombination at cer. Xer-cer is constrained to multimer resolution by accessory proteins ArgR and PepA. Recently, it has been shown that ArgR and PepA influence Cre-mediated recombination at a cer-lox hybrid site in vitro, defining the structure of the synaptic complex. We show here that both ArgR and PepA are required for stable maintenance of the P1 prophage. It is extremely difficult to establish P1 in a strain lacking PepA and the prophage was lost rapidly once selection was removed. ArgR plays a less crucial role although its absence significantly increased prophage loss. The effect of the accessory proteins is seen only at physiological concentrations of Cre; when the recombinase is expressed from a multicopy plasmid, the prophage is unstable even in the presence of ArgR and PepA. We propose that ArgR and PepA are involved in Cre-lox recombination in vivo, probably by constraining the system to resolution of prophage dimers.     

Control of Cre recombination by regulatory elements from Xer recombination systems

Site-specific recombination by the Cre recombinase takes place at a simple DNA site (loxP), requires no additional proteins and gives topologically simple recombination products. In contrast, cer and psi sites for Xer recombination contain approximately 150 bp of accessory sequences, require accessory proteins PepA, ArgR and ArcA, and the products are specifically linked to form a four-noded catenane. Here, we use hybrid sites consisting of accessory sequences of cer or psi fused to loxP to probe the function of accessory proteins in site-specific recombination. We show that PepA instructs Cre to produce four-noded catenane, but is not required for recombination at these hybrid sites. Mutants of Cre that require PepA and accessory sequences for efficient recombination were selected. PepA-dependent Cre gave products with a specific topology and displayed resolution selectivity. Our results reveal that PepA acts autonomously in the synapsis of psi and cer accessory sequences and is the main architectural element responsible for intertwining accessory site DNA. We suggest that accessory proteins can activate recombinases simply by synapsing the regulatory DNA sequences, thus bringing the recombination sites together with a specific geometry. This may occur without the need for protein-protein interactions between accessory proteins and the recombinases.     

The ArcA/ArcB two-component regulatory system of Escherichia coli is essential for Xer site-specific recombination at psi

Two recombinases, XerC and XerD, act at the recombination sites psi and cer in plasmids pSC101 and ColE1 respectively. Recombination at these sites maintains the plasmids in a monomeric state and helps to promote stable plasmid inheritance. The accessory protein PepA acts at both psi and cer to ensure that only intramolecular recombination takes place. An additional accessory protein, ArgR, is required for recombination at cer but not at psi . Here, we demonstrate that the ArcA/ArcB two-component regulatory system of Escherichia coli , which mediates adaptation to anaerobic growth conditions, is required for efficient recombination in vivo at psi . Phosphorylated ArcA binds to psi in vitro and increases the efficiency of recombination at this site. Binding of ArcA to psi may contribute to the formation of a higher order synaptic complex between a pair of psi sites, thus helping to ensure that recombination is intramolecular.     

Peptidase activity of Escherichia coli aminopeptidase A is not required for its role in Xer site-specific recombination

Xer site-specific recombination is required for the stable inheritance of multicopy plasmids and the normal segregation of the bacterial chromosome in Escherichia coli.Two related recombinases and two accessory proteins are essential for Xer-mediated recombination at cer, a recombination site in the plasmid ColE1 The accessory proteins, ArgR and PepA, function in ensuring that the Xer recombination reaction acts exclusively intramolecularly, converting plasmid dimers into monomers and not vice versa. PepA is an amino-exopeptidase, but its molecular role in the Xer recombination mechanism is unclear. Here we show that a mutation directed at the presumptive active site of PepA creates a protein with no detectable peptidase activity in vitro or in vivo, but which still functions normality in Xer site-specific recombination at cer.     

Xer-mediated site-specific recombination in vitro.

The Xer site-specific recombination system acts at ColE1 cer and pSC101 psi sites to ensure that these plasmids are in a monomeric state prior to cell division. We show that four proteins, ArgR, PepA, XerC and XerD are necessary and sufficient for recombination between directly repeated cer sites on a supercoiled plasmid in vitro. Only PepA, XerC and XerD are required for recombination at psi in vitro. Recombination at cer and psi in vitro requires negative supercoiling and is exclusively intramolecular. Strand exchange at cer produces Holliday junction-containing products in which only the top strands have been exchanged. This reaction requires the catalytic tyrosine residue of Xer C but not that of XerD. Recombination at psi gives catenated circular resolution products. Strand exchange at psi is sequential. XerC catalyses the first (top) strand exchange to make a Holiday junction intermediate and XerD catalyses the second (bottom) strand exchange.     

DNA binding of Escherichia coli arginine repressor mutants altered in oligomeric state

The Escherichia coli arginine repressor (ArgR) controls expression of the arginine biosynthetic genes and acts as an accessory protein in Xer site-specific recombination at cer and related plasmid recombination sites. The hexameric wild-type protein shows L -arginine-dependent DNA binding. In this work, ArgR mutants that are defective in trimer–trimer interactions and bind DNA as trimers in an L -arginine-independent manner are isolated and characterized. Whereas the wild-type ArgR hexamer exhibits high-affinity binding to two repeated ARG boxes separated by 3 bp (each ARG box containing two identical dyad symmetrical 9 bp half-sites), the trimeric mutants bind to and footprint three adjacent half-sites of this 'idealized' substrate. Trimeric ArgR is impaired in its ability to repress the arginine biosynthetic genes and in Xer site-specific recombination. In the absence of L -arginine, residual wild-type ArgR-binding occurs as trimers. The binding of an N-terminal 77-amino-acid DNA-binding domain to idealized ARG boxes is also characterized.     

Xer-mediated site-specific recombination at cer generates Holliday junctions in vivo.

Normal segregation of the Escherichia coli chromosome and stable inheritance of multicopy plasmids such as ColE1 requires the Xer site-specific recombination system. Two putative lambda integrase family recombinases, XerC and XerD, participate in the recombination reactions. We have constructed an E. coli strain in which the expression of xerC can be tightly regulated, thereby allowing the analysis of controlled recombination reactions in vivo. Xer-mediated recombination in this strain generates Holliday junction-containing DNA molecules in which a specific pair of strands has been exchanged in addition to complete recombinant products. This suggests that Xer site-specific recombination utilizes a strand exchange mechanism similar or identical to that of other members of the lambda integrase family of recombination systems. The controlled in vivo recombination reaction at cer requires recombinase and two accessory proteins, ArgR and PepA. Generation of Holliday junctions and recombinant products is equally efficient in RuvC- and RuvC+ cells, and in cells containing a multicopy RuvC+ plasmid. Controlled XerC expression is also used to analyse the efficiency of recombination between variant cer sites containing sequence alterations and heterologies within their central regions.     

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.     

Resolution of ColE1 dimers requires a DNA sequence implicated in the three-dimensional organization of the cer site.

Plasmid ColE1 specifies a recombination site (cer) which participates in the conversion of plasmid dimers to monomers. The uncontrolled accumulation of dimers (and higher oligomeric forms) would otherwise lead to plasmid instability. Exonuclease III-generated deletions have been used to define the left-hand boundary of the cer site. Deletions which have lost up to 60 bp adjacent to the boundary no longer mediate the conversion of plasmid dimers to monomers, but still recombine with a wild-type site. Although this boundary region is essential for dimer resolution, its DNA sequence is poorly conserved among multimer resolution sites in related plasmids. We present evidence that its function is to influence the three-dimensional organization of the site and suggest that it may be required for the formation of a condensed nucleoprotein complex.     

Synaptic intermediates in bacteriophage lambda site-specific recombination: integrase can align pairs of attachment sites.

Bacteriophage lambda uses site-specific recombination to move its DNA into and out of the Escherichia coli genome. The recombination event is mediated by the recombinase integrase (Int) together with several accessory proteins through short specific DNA sequences known as attachment sites. A gel mobility shift assay has been used to show that, in the absence of accessory proteins, Int can align and hold together two DNA molecules, each with an attachment site, to form stable non-covalent 'bimolecular complexes'. Each attachment site must have both core and arm binding sites for Int to participate in a bimolecular complex. These stable structures can be formed between pairs of attL and attP attachment sites, but cannot include attB or attR sites; they are inhibited by integration host factor (IHF) protein. The bimolecular complexes are shown to represent a synaptic intermediate in the reaction in which Int protein promotes the IHF-independent recombination of two attL sites. These complexes should enable a detailed analysis of synapsis for this pathway.     

Lipooligosaccharide biosynthesis in Neiseria gonorrhoeae: cloning, identification and characterization of the α1,5 heptosyltransferase I gene (rfaC)

The Escherichia coli arginine repressor (ArgR) is an l -arginine-dependent DNA-binding protein that controls expression of the arginine biosynthetic genes and is required as an accessory protein in Xer site-specific recombination at cer and related recombination sites in plasmids. Site-directed mutagenesis was used to isolate two mutants of E. coli ArgR that were defective in arginine binding. Results from in vivo and in vitro experiments demonstrate that these mutants still act as repressors and bind their specific DNA sequences in an arginine-independent manner. Both mutants support Xer site-specific recombination at cer. One of the mutant proteins was purified and shown to bind to its DNA target sequences in vitro with different affinity and as a different molecular species to wild-type ArgR.