Asymmetry of shufflon-specific recombination sites in plasmid R64 inhibits recombination between direct sfx sequences

The shufflon of plasmid R64 consists of four DNA segments separated and flanked by seven sfx recombination sites. Rci-mediated recombination between any inverted sfx sequences causes inversion of the DNA segments independently or in groups. The R64 shufflon selects one of seven pilV genes encoding type IV pilus adhesins, in which the N-terminal region is constant, while the C-terminal regions are variable. The R64 sfx sequences are asymmetric. The sfx central region and right arm sequences are conserved, but left arm sequences are not. Here we constructed a symmetric sfx sequence, in which the sfx left arm sequence was changed to the inverted repeat of the right arm sequence and made artificial shufflon segments carrying symmetric sfx sequences in inverted or direct orientations. The symmetric sfx sequence exhibited the highest inversion frequency in a shufflon segment flanked by two inverted sfx sequences. Rci-dependent deletion of a shufflon segment flanked by two direct symmetric sfx sequences was observed, suggesting that asymmetry of R64 sfx sequences inhibits recombination between direct sfx sequences. In addition, intermolecular recombination between symmetric sfx sequences was also observed. The extra C-terminal domain of Rci was shown to be essential for inversion of the R64 shufflon using asymmetric sfx sequences but not essential for recombination using symmetric sfx sequences, suggesting that the Rci C-terminal segment helps the binding of Rci to asymmetric sfx sequences. Rci protein lacking the C-terminal domain bound to both arms of symmetric sfx sequence but only to the right arm of asymmetric sfx sequence.     

Purification and characterization of the R64 shufflon-specific recombinase

The shufflon, a multiple DNA inversion system in plasmid R64, consists of four invertible DNA segments which are separated and flanked by seven 19-bp repeat sequences. The product of a site-specific recombinase gene, rci, promotes site-specific recombination between any two of the inverted 19-bp repeat sequences of the shufflon. To analyze the molecular mechanism of this recombination reaction, Rci protein was overproduced and purified. The purified Rci protein promoted the in vitro recombination reaction between the inverted 19-bp repeats of supercoiled DNA of a plasmid carrying segment A of the R64 shufflon. The recombination reaction was enhanced by the bacterial host factor HU. Gel electrophoretic analysis indicated that the Rci protein specifically binds to the DNA segments carrying the 19-bp sequences. The binding affinity of the Rci protein to the four shufflon segments as well as four synthetic 19-bp sequences differed greatly: among the four 19-bp repeat sequences, the repeat-a and -d sequences displayed higher affinity to Rci protein. These results suggest that the differences in the affinity of Rci protein for the 19-bp repeat sequences determine the inversion frequencies of the four segments.     

Specific binding of the NikA protein to one arm of 17-base-pair inverted repeat sequences within the oriT region of plasmid R64.

Products of the nikA and nikB genes of plasmid R64 have been shown to form a relaxation complex with R64 oriT DNA and to function together as an oriT-specific nickase. We purified the protein product of the nikA gene. The purified NikA protein bound specifically to the oriT region of R64 DNA. Gel retardation assays and DNase I footprinting analyses indicated that the NikA protein bound only to the right arm of 17-bp inverted repeat sequences; the right arm differed from the left arm by a single nucleotide. The binding site is proximal to the nick site and within the 44-bp oriT core sequence. Binding of the NikA protein induced DNA bending within the R64 oriT sequence.     

Plasmids carrying cloned fragments of RF DNA from the filamentous phage φLf can be integrated into the host chromosome via site-specific integration and homologous recombination

Different regions of RF DNA from the filamentous bacteriophage phiLf were cloned in Escherichia coli vectors that can not be maintained in Xanthomonas. After introduction into X. campestris pv. campestris 17 (Xc17), most of these constructs were found to integrate into the host chromosome, either by recA-dependent homologous recombination or recA-independent site-specific integration. Mutations in himA, which codes for the alpha-subunit of the Integration Host Factor, does not affect the integration. Integration occurs into a chromosomal region which harbors a copy of a defective phage (4445 bp) that shares a high degree of identity with the phiLf genome. While various parts of the 4445-bp region are susceptible to homologous recombination, site-specific integration requires the attB sequence on the chromosome and the phage attP. The attB shows a high level of sequence identity (22 out of 28 bp) to the dif site required for E. coli Xer site-specific recombination, including the 6-bp central region, and 8/11 identity in both the left XerC-binding arm and the right XerD-binding arm, with the innermost 5 nt of the arms forming a dyad symmetry that is also present in dif. The attP has the same central region and shows 10/11 identity to the dif site in the left arm, but the sequence of the right arm is less conserved than that of attB. The smallest regions still capable of mediating integration are a cloned 72-bp phiLf attP-containing sequence and a 51-bp Xc17 attB-containing sequence, which was reinserted into the Xc17 chromosome after the 4445-bp region had been deleted, indicating that accessory sequences are not necessary and that the integrase required for site-specific integration is neither specified by the 4445-bp Xc17 chromosomal region nor encoded by the phiLf genome.     

Transfer RNA genes frequently serve as integration sites for prokaryotic genetic elements.

The DNA sequences were determined at the boundaries of the integrated copy of the archaebacterial genetic element SSV1. A 44 bp sequence present as a single copy on the 15.5 kb circular SSV1 DNA flanked the integrated copy as a direct DNA sequence repeat, suggesting that SSV1 integration occurred by recombination between this 44 bp SSV1 sequence and an identical sequence on the bacterial chromosome. At the left attachment site, a region encompassing the 44 bp attachment core sequence and the 31 nucleotides upstream of it displayed all characteristics expected for an arginine tRNA gene. An analysis of published attachment site sequences of other systems revealed that tRNA genes also constitute the bacterial attachment site in the case of three temperate phages and two transmissible plasmids in eubacteria, indicating a widespread occurrence of tRNA genes as integration target sites. This finding may be important for the understanding of mechanisms and evolution of site-specific recombination.     

Cloning and nucleotide sequence of the ColIb shufflon

The R64 shufflon is a novel type of DNA rearrangement in which four DNA segments invert independently or in groups. The related plasmid ColIb carries a variant shufflon. The present sequence analysis shows that the ColIb shufflon consists of three DNA segments that are highly homologous to the A, B, and C segments of the R64 shufflon. The 329-bp D segment of R64 is not present in the ColIb shufflon. As in the case of R64, the ColIb shufflon may act as a biological switch to select one of the six open reading frames in which the N-terminal region is constant while the C-terminal region is variable.     

Structure and function of 59-base element recombination sites associated with mobile gene cassettes

The integration of gene cassettes into integrons is effected by site-specific recombination catalysed by an integrase, IntI, encoded by the integron. The cassette-associated recombination sites, 59-base elements, are not highly conserved and vary in length from 57 to 141 bp. They can be identified by their location and the relationship of over 20 bp at their outer ends to consensus sequences that are imperfect inverted repeats of one another. The recombination cross-over occurs close to one end of the 59-base element, within a conserved core site with the consensus sequence GTTAGGC or GTTRRRY. By introducing single-base changes at each of these positions in the aadB 59-base element, bases that are critical for site activity were identified. The recombination cross-over was also localized to a unique position between the adjacent G and T residues. Changes introduced in the conserved AAC of the inverse core site (GCCTAAC or RYYYAAC) located at the opposite end of the 59-base element also reduced site activity but to a lesser extent. Sequences of rare recombinants revealed an alternative position for strand exchange and led to the conclusion that 59-base elements comprise two simple sites, analogous to those recognized by other integrases, with each simple site made up of a pair of inversely oriented IntI binding domains separated by a spacer of 7 or 8 bp. Re-examination of the sequences of all known 59-base elements revealed that this simple site configuration was present at both the left and right ends in all 59-base elements. The identity of bases in the spacer is not required for efficient recombination and the cross-over is located at one end of the spacer, suggesting that during IntI1-mediated recombination only one strand exchange occurs.     

Interaction of int protein with specific sites on lambda att DNA.

We have studied the interaction of highly purified Int protein with DNA restriction fragments from the lambda phage attachment site (attP) region. Two different DNA sequences are protected by bound Int protein against partial digestion by either pancreatic DNAase or neocarzinostatin. One Int binding site includes the 15 bp common core sequence (the crossover region for site-specific recombination) plus several bases of sequence adjoining the core in both the P and P' arms. The second Int-protected site occurs 70 bp to the right of the common core in the P' arm, just at the distal end of the sequence encoding Int protein. The two Int binding sites are of comparable size, 30-35 bp, but do not share any extensive sequence homology. The interaction of Int with the two sites is distinctly different, as defined by the observation that only the site in the P' arm and not the site at the common core region is protected by Int in the face of challenge by the polyanion heparin. Restriction fragments containing DNA from the bacterial attachment site (attB) region exhibit a different pattern of interaction with Int. In the absence of heparin, a smaller (15 bp) sequence, which includes the left half of the common core region and the common core-B arm juncture, is protected against nuclease digestion by Int protein. No sequences from this region are protected by Int in the presence of heparin.     

Nucleotide sequence of the R721 shufflon.

The shufflon is a DNA region that undergoes complex rearrangement mediated by the product of a putative site-specific recombinase gene, rci. The DNA sequences of the shufflon region and the rci gene of IncI2 plasmid R721 were determined. The R721 shufflon consists of three invertible DNA segments that are homologous to the shufflon segments found in IncI1 plasmid R64. Structural analysis of open reading frames indicated that the R721 shufflon possibly functions as a biological switch for selecting one of the six pilV genes in which the N-terminal region is constant and the C-terminal region is variable. The R721 rci gene was shown to encode a basic protein of 374 amino acid residues.     

Mating variation by DNA inversions of shufflon in plasmid R64

Gene organization of the 54-kb transfer region of IncI1 plasmid R64 was deduced from the DNA sequence. Forty-eight ORFs were found in this region. A unique DNA rearrangement designated shufflon is located at the downstream region of an operon responsible for synthesis of thin pilus. The shufflon of R64 consists of four DNA segments, designated as A, B, C, and D, which are flanked and separated by seven 19-bp repeat sequences. Site-specific recombination mediated by the product of the rci gene between any two inverted repeats results in a complex DNA rearrangement. An analysis of open reading frames revealed that the shufflon is a biological switch to select one of seven C-terminal segments of the pilV genes. The products of pilV genes were shown to be components of thin pilus which was required for liquid mating.Seven R64 derivatives where the pilV genes were fixed in the seven C-terminal segments were constructed and their transfer frequencies in liquid mating were measured using various bacterial strains as recipients. Transfer frequencies of R64 in liquid mating strongly depended on the combination of C-terminal segments of the pilV genes in donor cells and bacterial strains of recipient cells, suggesting that the shufflon determines the recipient specificity in liquid mating of plasmid R64.     

DNA sequence of the att region of coliphage 434

Phages lambda and 434 are related phages that insert at the same site on the Escherichia coli chromosome. A 5.9-kb SalI-BamHI fragment derived from phage 434 was shown to hybridize to a 0.5-kb probe carrying attP-lambda. A 0.8-kb Bam HI-TaqI fragment subcloned into pBR327 was used for sequencing. The sequence of the 500 bp around the insertion site is given here, Comparison of the lambda and 434 sequence shows that the following regions are conserved: the coding sequence for the integrase protein (only 162 bp have been sequenced corresponding to the carboxy terminus), the 15-bp common core at the insertion site, and the three integrase-binding sites flanking the insertion site. The lambda and 434 sequences diverge radically to the left of base-197, suggesting that DNA to the left of that point plays no specific role in insertion or its regulation.     

Mutational analysis of the R64 oriT region: requirement for precise location of the NikA-binding sequence.

Conjugative DNA transfer of IncI1 plasmid R64 is initiated by the introduction of a site- and strand-specific nick into the origin of transfer (oriT). In R64 oriT, 17-bp (repeat A and B) and 8-bp inverted-repeat sequences with mismatches are located 8 bp away from the nick site. The nicking is mediated by R64 NikA and NikB proteins. To analyze the functional organization of the R64 oriT region, various deletion, insertion, and substitution mutations were introduced into a 92-bp minimal R64 oriT sequence and their effects on oriT function were investigated. This detailed analysis confirms our previous prediction that the R64 oriT region consists of an oriT core sequence and additional sequences necessary for full oriT activity. The oriT core sequence consists of the repeat A sequence, which is recognized by R64 NikA protein, and the nick region sequence, which is conserved among various origins of transfer and is most probably recognized by NikB protein. The oriT core sequence is sufficient for NikAB-mediated oriT-specific nicking. Furthermore, it was shown that the repeat A sequence is essential for localization to a precise position relative to the nick site for oriT function. This seems to be required for the formation of a functional ternary complex consisting of NikA and NikB proteins and oriT DNA. The repeat B sequence and 8-bp inverted repeat sequences are suggested to be required for the termination of DNA transfer.     

Extent of the DNA sequence required in integration of staphylococcal bacteriophage L54a.

We characterized the minimum length of the DNA sequence of the attachment sites involved in the integrative recombination of staphylococcal bacteriophage L54a. A DNA fragment carrying the functional viral attachment site (attP) or the bacterial attachment site (attB) was sequentially trimmed, recloned, and tested for integrative recombination in vivo. The size of the functional attP site was at least 228 base pairs (bp) but no more than 235 bp. The left endpoint of the attP site was located to between positions -142 and -140, whereas the right endpoint was located to between positions +86 and +93 with respect to the center of the core sequence. The attB site was located to within a 27-bp sequence, from position -15 to +12, which included the 18-bp core sequence.     

Short-range and long-range context effects on coliphage T4 endonuclease II-dependent restriction.

Synthetic sites inserted into a plasmid were used to analyze the sequence requirements for in vivo DNA cleavage dependent on bacteriophage T4 endonuclease II. A 16-bp variable sequence surrounding the cleavage site was sufficient for cleavage, although context both within and around this sequence influenced cleavage efficiency. The most efficiently cleaved sites matched the sequence CGRCCGCNTTGGCNGC, in which the strongly conserved bases to the left were essential for cleavage. The less-conserved bases in the center and in the right half determined cleavage efficiency in a manner not directly correlated with the apparent base preference at each position; a sequence carrying, in each of the 16 positions, the base most preferred in natural sites in pBR322 was cleaved infrequently. This, along with the effects of substitutions at one or two of the less-conserved positions, suggests that several combinations of bases can fulfill the requirements for recognition of the right part of this sequence. The replacements that improve cleavage frequency are predicted to influence helical twist and roll, suggesting that recognition of sequence-dependent DNA structure and recognition of specific bases are both important. Upon introduction of a synthetic site, cleavage at natural sites within 800 to 1,500 bp from the synthetic site was significantly reduced. This suggests that the enzyme may engage more DNA than its cleavage site and cleaves the best site within this region. Cleavage frequency at sites which do not conform closely to the consensus is, therefore, highly context dependent. Models and possible biological implications of these findings are discussed.     

NikAB- or NikB-dependent intracellular recombination between tandemly repeated oriT sequences of plasmid R64 in plasmid or single-stranded phage vectors

The origin of transfer (oriT) of a bacterial plasmid plays a key role in both the initiation and termination of conjugative DNA transfer. We have previously shown that a conjugation-dependent recombination between the tandem R64 oriT sequences cloned into pHSG398 occurred, resulting in the deletion of the intervening sequence during DNA transfer. In this study, we tandemly cloned two oriT sequences of IncI1 plasmid R64 into pUC18. Specific recombination between the two oriT sequences in pUC18 was observed within Escherichia coli cells harboring mini-R64. This recombination was found to be independent of both the recA gene and conjugative DNA transfer. The R64 genes nikA and nikB, required for conjugal DNA processing, were essential for this recombination. Although a fully active 92-bp oriT sequence was required at one site for the recombination, the 44-bp oriT core sequence was sufficient at the other site. Furthermore, when two oriT sequences were tandemly cloned into the single-stranded phage vector M13 and propagated within E. coli cells, recombination between the two oriT sequences was observed, depending on the nikB gene. These results suggest that the R64 relaxase protein NikB can execute cleavage and rejoining of single-stranded oriT DNA within E. coli cells, whereas such a reaction in double-stranded oriT DNA requires collaboration of the two relaxosome proteins, NikA and NikB.     

DNA from the A6S/2 crown gall tumor contains scrambled Ti-plasmid sequences near its junctions with plant DNA

The A6S/2 tumor incited on tobacco by Agrobacterium tumefaciens harboring the octopine-type A6 Ti plasmid contains one insert of Ti-plasmid sequences (the T DNA). This 13 kb insert is derived from a colinear sequence in the Ti plasmid (the T region) and becomes attached to plant DNA in the nucleus of the host cell. We have determined the DNA sequence encompassing the left end of the T region of the A6 Ti plasmid and the corresponding portion of the A6S/2 T DNA. The two sequences are identical for at least 806 bp. To the left of the divergence point, the tumor contains five partially overlapping sequences that are direct or inverted repeats of sequences to the right of the divergence point. The Ti plasmid contains only the right member of each of these repeats. We have also performed heteroduplex studies that indicate that this T DNA has a 520 bp inverted repeat of an internal sequence at the right end near its junction with plant DNA. The repeated sequences near the ends of the T DNA resemble the repeats of adenovirus type 12 sequences found near its junction with host DNA. We discuss data suggesting that the 23 bp to the immediate right of the divergence point of the A6 left junction form a site important in some step in the transfer of T-region DNA from the bacteria to the plant.     

Dissection of the bacteriophage Mu strong gyrase site (SGS): significance of the SGS right arm in Mu biology and DNA gyrase mechanism

The bacteriophage Mu strong gyrase site (SGS), required for efficient phage DNA replication, differs from other gyrase sites in the efficiency of gyrase binding coupled with a highly processive supercoiling activity. Genetic studies have implicated the right arm of the SGS as a key structural feature for promoting rapid Mu replication. Here, we show that deletion of the distal portion of the right arm abolishes efficient binding, cleavage, and supercoiling by DNA gyrase in vitro. DNase I footprinting analysis of the intact SGS revealed an adenylyl imidodiphosphate-dependent change in protection in the right arm, indicating that this arm likely forms the T segment that is passed through the cleaved G segment during the supercoiling reaction. Furthermore, in an SGS derivative with an altered right-arm sequence, the left arm showed these changes, suggesting that the selection of a T segment by gyrase is determined primarily by the sequences of the arms. Analysis of the sequences of the SGS and other gyrase sites suggests that the choice of T segment correlates with which arm possesses the more extensive set of phased anisotropic bending signals, with the Mu right arm possessing an unusually extended set of such signals. The implications of these observations for the structure of the gyrase-DNA complex and for the biological function of the Mu SGS are discussed.     

Genetic analysis of the strong gyrase site (SGS) of bacteriophage Mu: localization of determinants required for promoting Mu replication

The Mu strong gyrase site (SGS), located in the centre of the Mu genome, is required for efficient Mu replication, as it promotes synapsis of the prophage termini. Other gyrase sites tested, even very strong ones, were unable to substitute for the SGS in Mu replication. To determine the features required for its unique properties, a deletion analysis was performed on the SGS. For this analysis, we defined the 20 bp centred on the midpoint of the 4 bp staggered cleavage made by gyrase to be the 'core' and the flanking sequences to be the 'arms'. The deletion analysis showed that (i) approximately 40 bp of the right arm is required, in addition to core sequences, for both efficient Mu replication and gyrase cleavage; and (ii) the left arm was not required for efficient Mu replication, although it was required for efficient gyrase cleavage. These observations implicated the right arm as the unique feature of the SGS. The second observation showed that strong gyrase cleavage and Mu replication could be dissociated and suggested that even weak gyrase sites, if supplied with the right arm of the SGS, could promote Mu replication. Hybrid sites were constructed with gyrase sites that could not support efficient Mu replication. The SGS right arm was used to replace one arm of the strong pSC101 gyrase site or the weaker pBR322 site. The pSC101 hybrid site allowed efficient Mu replication, whereas the pBR322 hybrid site allowed substantial, but reduced, replication. Hence, it appears that optimal Mu replication requires a central strong gyrase site with the properties imparted by the right arm sequences. Possible roles for the SGS right arm in Mu replication are addressed.     

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.     

Structural bases of a long-stretched deletion: completing the lambda plac5 DNA primary structure.

In studying molecular mechanisms of specialised transduction, the lacI (E. coli)-Ea47 (lambda) DNA junction in transducing bacteriophage lambda plac 5 has been structurally elucidated, thus yielding the complete sequence of lambda plac 5 DNA including the lac5 substitution, a well-known segment of lambdoid vectors. The lambda plac5 DNA is shown to consist of 19368 bp (lambda left arm) + 3924 bp (lac5 substitution) + 25353 bp (lambda right arm), totally amounting to 48645 bp. The presence of the phage rho bL promoter near to the right end of the lac5 insert is shown. The lacI gene distal end in lambda plac5 proved to be much longer than it was postulated earlier, coding for 224 C-terminal amino acid residues of lac repressor. Both the recombination studied in this paper and the earlier studied abnormal prophage excision (2, 3) occur near to Chi-like structures (chi*lacI and chi*lom, respectively). On the basis of the data obtained, a key role of the E. coli RecBCD system and Chi-like sequences in the formation of deletions in bacterial cells is suggested.