HK022 bacteriophage Integrase mediated RMCE as a potential tool for human gene therapy.

HK022 coliphage site-specific recombinase Integrase (Int) can catalyze integrative site-specific recombination and recombinase-mediated cassette exchange (RMCE) reactions in mammalian cell cultures. Owing to the promiscuity of the 7 bp overlap sequence in its att sites, active 'attB' sites flanking human deleterious mutations were previously identified that may serve as substrates for RMCE reactions for future potential gene therapy. However, the wild type Int proved inefficient in catalyzing such RMCE reactions. To address this low efficiency, variants of Int were constructed and examined by integrative site-specific recombination and RMCE assays in human cells using native 'attB' sites. As a proof of concept, various Int derivatives have demonstrated successful RMCE reactions using a pair of native 'attB' sites that were inserted as a substrate into the human genome. Moreover, successful RMCE reactions were demonstrated in native locations of the human CTNS and DMD genes whose mutations are responsible for Cystinosis and Duchene Muscular Dystrophy diseases, respectively. This work provides a steppingstone for potential downstream therapeutic applications.

Probing the structure of Nun transcription arrest factor bound to RNA polymerase.

The coliphage HK022 protein Nun transcription elongation arrest factor inhibits RNA polymerase translocation. In vivo, Nun acts specifically to block transcription of the coliphage λ chromosome. Using in vitro assays, we demonstrate that Nun cross-links RNA in an RNA:DNA hybrid within a ternary elongation complex (TEC). Both the 5' and the 3' ends of the RNA cross-link Nun, implying that Nun contacts RNA polymerase both at the upstream edge of the RNA:DNA hybrid and in the vicinity of the catalytic center. This finding suggests that Nun may inhibit translocation by more than one mechanism. Transcription elongation factor GreA efficiently blocked Nun cross-linking to the 3' end of the transcript, whereas the highly homologous GreB factor did not. Surprisingly, both factors strongly suppressed Nun cross-linking to the 5' end of the RNA, suggesting that GreA and GreB can enter the RNA exit channel as well as the secondary channel, where they are known to bind. These findings extend the known action mechanism for these ubiquitous cellular factors.

Cancer-specific binary expression system activated in mice by bacteriophage HK022 Integrase.

Binary systems based on site-specific recombination have been used for tumor specific transcription targeting of suicide genes in animal models. In these binary systems a site specific recombinase or integrase that is expressed from a tumor specific promoter drives tumor specific expression of a cytotoxic gene. In the present study we developed a new cancer specific binary expression system activated by the Integrase (Int) of the lambdoid phage HK022. We demonstrate the validity of this system by the specific expression of a luciferase (luc) reporter in human embryonic kidney 293T (HEK293T) cells and in a lung cancer mouse model. Due to the absence viral vectors and of cytotoxicity the Int based binary system offers advantages over previously described counterparts and may therefore be developed into a safer cancer cell killing system.

Efficient Flp-Int HK022 dual RMCE in mammalian cells.

Recombinase-mediated cassette exchange, or RMCE, is a clean approach of gene delivery into a desired chromosomal location, as it is able to insert only the required sequences, leaving behind the unwanted ones. RMCE can be mediated by a single site-specific DNA recombinase or by two recombinases with different target specificities (dual RMCE). Recently, using the Flp-Cre recombinase pair, dual RMCE proved to be efficient, provided the relative ratio of the enzymes during the reaction is optimal. In the present report, we analyzed how the efficiency of dual RMCE mediated by the Flp-Int (HK022) pair depends on the variable input of the recombinases-the amount of the recombinase expression vectors added at transfection-and on the order of the addition of these vectors: sequential or simultaneous. We found that both in the sequential and the simultaneous modes, the efficiency of dual RMCE was critically dependent on the absolute and the relative concentrations of the Flp and Int expression vectors. Under optimal conditions, the efficiency of 'simultaneous' dual RMCE reached ∼12% of the transfected cells. Our results underline the importance of fine-tuning the reaction conditions for achieving the highest levels of dual RMCE.

Arm site independence of coliphage HK022 integrase in human cells.

The integrase encoded by the lambdoid phage HK022 (Int-HK022) resembles its coliphage λ counterpart (Int-λ) in the roles of the cognate DNA arm binding sites and in controlling the direction of the reaction. We show here that within mammalian cells, Int-HK022 does not exhibit such a control. Rather, Int-HK022 recombined between all ten possible pairwise att site combinations, including attB × attB that was more effective than the conventional integrative attP × attB reaction. We further show that Int-HK022 depends on the accessory integration host factor (IHF) protein considerably less than Int-λ and exhibits stronger binding affinity to the att core. These differences explain why wild-type Int-HK022 is active in mammalian cells whereas Int-λ is active there only as an IHF-independent mutant.

Site-specific recombination in the cyanobacterium Anabaena sp. strain PCC 7120 catalyzed by the integrase of coliphage HK022.

The integrase (Int) of the lambda-like coliphage HK022 catalyzes the site-specific integration and excision of the phage DNA into and from the chromosome of its host, Escherichia coli. Int recognizes two different pairs of recombining sites attP x attB and attL x attR for integration and excision, respectively. This system was adapted to the cyanobacterium Anabaena sp. strain PCC 7120 as a potential tool for site-specific gene manipulations in the cyanobacterium. Two plasmids were consecutively cointroduced by conjugation into Anabaena cells, one plasmid that expresses HK022 Int recombinase and the other plasmid that carries the excision substrate P(glnA)-attL-T1/T2-attR-lacZ, where T1/T2 are the strong transcription terminators of rrnB, to prevent expression of the lacZ reporter under the constitutive promoter P(glnA). The Int-catalyzed site-specific recombination reaction was monitored by the expression of lacZ emanating as a result of T1/T2 excision. Int catalyzed the site-specific excision reaction in Anabaena cells when its substrate was located either on the plasmid or on the chromosome with no need to supply an accessory protein, such as integration host factor and excisionase (Xis), which are indispensable for this reaction in its host, E. coli.

Optimization of coliphage HK022 Integrase activity in human cells.

The Integrase (Int) site-specific recombinase of coliphage HK022 catalyzes integrative and excisive DNA recombination between two attachment (att) sites in human cells without the need to supply the accessory proteins Integration Host Factor (IHF) and Excisionase (Xis). Previous work has shown that under these conditions, reactions in cis, i.e. both att sites are located on the same chromosome, can be detected without selection. However, recombination in trans, i.e. one att site positioned on a chromosome and the other on an episomal vector, was detected only after selection. Here we show that optimization of the int-HK022 gene for human codon usage according to the GeneOptimizer software algorithm, as well as addition of accessory proteins IHF and Xis improve the recombination efficiencies in human cells, such that recombinants in a trans reaction could be detected without selection.

Molecular analysis of recombinase-mediated cassette exchange reactions catalyzed by integrase of coliphage HK022.

The integrase (Int) protein of coliphage HK022 can catalyze in Escherichia coli as well as in in vitro integrative and excisive recombinase-mediated cassette exchange reactions between plasmids as substrates. Atomic force microscopy images have revealed that in the protein-DNA complexes that are formed, the plasmid substrates are connected via one and not two pairs of attachment sites. This observation, together with the elucidation of intermediate co-integrates between the two circular plasmids, suggest that a sequential mechanism of the RMCE reaction is possible.

Inhibition of a transcriptional pause by RNA anchoring to RNA polymerase.

We describe a mechanism by which nascent RNA inhibits transcriptional pausing. PutL RNA of bacteriophage HK022 suppresses transcription termination at downstream terminators and pausing within a nearby U-rich sequence. In vitro transcription and footprinting assays reveal that this pausing results from backtracking of RNA polymerase and that binding of nascent putL RNA to polymerase limits backtracking by restricting re-entry of the transcript into the RNA exit channel. The restriction is local and relaxes as the transcript elongates. Our results suggest that putL RNA binds to the surface of polymerase close to the RNA exit channel, a region that includes amino acid residues important for antitermination. Although binding is essential for antipausing and antitermination, these two activities of put differ: antipausing is limited to the immediate vicinity of the putL site, but antitermination is not. We propose that RNA anchoring to the elongation complex is a widespread mechanism of pause regulation.

Overexpression of phage HK022 Nun protein is toxic for Escherichia coli.

The Nun protein of coliphage HK022 excludes superinfecting lambda phage. Nun recognizes and binds to the N utilization (nut) sites on phage lambda nascent RNA and induces transcription termination. Overexpression of Nun from a high-copy plasmid is toxic for Escherichia coli, despite the fact that nut sites are not encoded in the E. coli genome. Cells expressing Nun cannot exit stationary phase. Toxicity is related to transcription termination, since host and nun mutations that block termination also suppress cell killing. Nun inhibits expression of wild-type lacZ, but not lacZ expressed from the Crp/cAMP-independent lacUV5 promoter. Microarray and proteomic analyses show that Nun down-regulates crp and tnaA. Crp overexpression and high indole concentrations partially reverse Nun-mediated toxicity and restore lacZ expression.

The Y39A mutation of HK022 Nun disrupts a boxB interaction but preserves termination activity.

Coliphage HK022 Nun protein targets phage lambda nut boxB RNA and acts as a transcriptional terminator, counteracting the phage lambda N protein, a suppressor of transcription termination. Both Nun and N protein interact directly with RNA polymerase, and Nun competes with N protein for boxB binding and prevents superinfection of Escherichia coli HK022 lysogens by lambda. Interaction of Trp18 of lambda N and A7 of boxB RNA in the N- boxB complex is essential for efficient antitermination. We found that the corresponding Nun mutation, Nun Y39A, disrupts the interaction between the aromatic ring of Y39 and A7, but the mutant retains in vivo termination activity. Stabilization of the complex by interaction of A7 with an aromatic amino acid is thus less important for Nun activity than it is for N activity. Structural investigations show similar binding of mutant and wild-type (wt) Nun protein to boxB RNA. The dissociation constants of the wt Nun(20-44)- boxB and mutant Nun(20-44)- boxB complex as well as the structures of the boxB RNA in both complexes are identical.

In vitro reconstitution and substrate specificity of a lantibiotic protease.

Lacticin 481 is a lanthionine-containing bacteriocin (lantibiotic) produced by Lactococcus lactis subsp. lactis. The final steps of lacticin 481 biosynthesis are proteolytic removal of an N-terminal leader sequence from the prepeptide LctA and export of the mature lantibiotic. Both proteolysis and secretion are performed by the dedicated ATP-binding cassette (ABC) transporter LctT. LctT belongs to the family of AMS (ABC transporter maturation and secretion) proteins whose prepeptide substrates share a conserved double-glycine type cleavage site. The in vitro activity of a lantibiotic protease has not yet been characterized. This study reports the purification and in vitro activity of the N-terminal protease domain of LctT (LctT150), and its use for the in vitro production of lacticin 481. The G(-2)A(-1) cleavage site and several other conserved amino acid residues in the leader peptide were targeted by site-directed mutagenesis to probe the substrate specificity of LctT as well as shed light upon the role of these conserved residues in lantibiotic biosynthesis. His 10-LctT150 did not process most variants of the double glycine motif and processed mutants of Glu-8 only very slowly. Furthermore, incorporation of helix-breaking residues in the leader peptide resulted in greatly decreased proteolytic activity by His 10-LctT150. On the other hand, His 10-LctT150 accepted all peptides containing mutations in the propeptide or at nonconserved positions of LctA. In addition, the protease domain of LctT was investigated by site-directed mutagenesis of the conserved residues Cys12, His90, and Asp106. The proteolytic activities of the resulting mutant proteins are consistent with a cysteine protease.

Phosphorylation of the integrase protein of coliphage HK022.

The integrase (Int) proteins of coliphages HK022 and lambda, are phosphorylated in one or more of their tyrosine residues. In Int of HK022 the phosphorylated residue(s) belong to its core-binding/catalytic domains. Wzc, a protein tyrosine kinase of Escherichia coli, is not required for Int phosphorylation in vivo, however, it can transphosphorylate the conserved Tyr(342) catalytic residue of Int in vitro. Int purified from cells that overexpress Wzc has a reduced activity in vitro. In vivo, the lysogenization of wild type HK022 as well as of lambda is not affected by the overexpression of Wzc. However, the nin5 mutant of lambda, which lacks a protein-tyrosine phosphatase gene, shows a significantly reduced lysogenization. It is suggested that phosphorylation of Int by Wzc down regulates the activity of Int.

Human genomic site-specific recombination catalyzed by coliphage HK022 integrase.

It has been previously demonstrated that the wild type integrase (Int) protein of coliphage HK022 can catalyze site-specific recombination in human cells between attachment (att) sites that were placed on extrachromosomal plasmids. In the present report it is shown that Int can catalyze the site-specific recombination reactions in a human cell culture on the chromosomal level. These include integrative (attP x attB) as well as excisive (attL x attR) reactions each in two configurations. In the cis configuration both sites are on the same chromosome, in the trans configuration one site is on a chromosome and the other on an episome. The reactions in cis were observed without any selection force, using the green fluorescent protein (GFP) as a reporter. The reactions in trans could be detected only when a selection force was applied, using the hygromycin-resistant (Hyg(R)) phenotype as a selective marker. All reactions were catalyzed without the need to supply any of the accessory proteins that are required by Int in its Escherichia coli host. The versatility of the att sites may be an advantage in the utilization of Int to integrate plasmid DNA into the genome, followed by a partial exclusion of the integrated plasmid.

Protection of antiterminator RNA by the transcript elongation complex.

Nascent transcripts encoded by the putL and putR sites of phage HK022 bind the transcript elongation complex and suppress termination at downstream transcription terminators. We report here that the chemical stability of putL RNA is considerably greater than that of the typical Escherichia coli message because the elongation complex protects this RNA from degradation. When binding to the elongation complex was prevented by mutation of either putL or RNA polymerase, RNA stability decreased more than 50-fold. The functional modification conferred by putL RNA on the elongation complex is also long-lived: the efficiency of terminator suppression remained high for at least 10 kb from the putL site. We find that RNase III rapidly and efficiently cleaved the transcript just downstream of the putL sequences, but such cleavage changed neither the stability of putL RNA nor the efficiency of antitermination. These results argue that the continuity of the RNA that connects put sequences to the growing point is not required for persistence of the antiterminating modification in vivo.

Role of E.coli NusA in phage HK022 Nun-mediated transcription termination.

The 109 amino acid residue Nun protein expressed from prophage HK022 excludes superinfecting phage lambda by arresting transcription on the lambda chromosome near the lambdanut sites. In vitro, the Nun N terminus binds to nascent lambdanutRNA, whereas the C terminus interacts with RNA polymerase and DNA template. Escherichia coli host factors, NusA, NusB, NusE (S10), and NusG, stimulate Nun-arrest. NusA binds the Nun C terminus and enhances formation of the Nun-nutRNA complex. Because of these in vitro activities of NusA, and since a nusA mutation (nusAE136K) blocked Nun in vivo, we assumed that NusA was required for Nun activity. However, using a nusAts strain, we find that NusA is required for termination at nutR but not at nutL. Furthermore, nusAE136K is dominant to nusA(+) for Nun-arrest, both in vitro and in vivo. NusAE136K shows increased affinity for Nun and, unlike NusA(+), can readily be recovered in a ternary complex with Nun and nutRNA. We propose NusAE136K suppresses Nun-arrest when it is a component of the transcription elongation complex, perhaps, in part, by blocking interactions between the Nun C terminus and RNA polymerase and DNA. We also find that in contrast to Nun-arrest, antitermination by lambda N requires NusA.

Site-specific recombination in Arabidopsis plants promoted by the Integrase protein of coliphage HK022.

The gene encoding the wild type Integrase protein of coliphage HK022 was integrated chromosomally and expressed in Arabidopsis thaliana plants. Double-transgenic plants cloned with the int gene as well as with a T-DNA fragment carrying the proper att sites in a tandem orientation showed that Int catalyzed a site-specific integration reaction (attP x attB) as well as a site-specific excision reaction (attL x attR). The reactions took place without the need to provide any of the accessory proteins that are required by Int in the bacterial host. When expressed in tobacco plants a GFP-Int fusion exhibits a predominant nuclear localization.

Transcription termination by phage HK022 Nun is facilitated by COOH-terminal lysine residues.

The 109-amino acid Nun protein of prophage HK022 excludes superinfecting bacteriophage lambda by blocking transcription elongation on the lambda chromosome. Multiple interactions between Nun and the transcription elongation complex are involved in this reaction. The Nun NH(2)-terminal arginine-rich motif binds BOXB sequence in nascent lambda transcripts, whereas the COOH terminus binds RNA polymerase and contacts DNA template. Nun Trp(108) is required for interaction with DNA and transcription arrest. We analyzed the role of the adjacent Lys(106) and Lys(107) residues in the Nun reaction. Substitution of the lysine residues with arginine (K106R/K107R) had no effect on transcription arrest in vitro or in vivo. Nun K106A/K107A was partially active, whereas Nun K106D/K107D was defective in vitro and failed to exclude lambda. All mutants bound RNA polymerase and BOXB. In contrast to Nun K106R/K107R and K106A/K107A, Nun K106D/K107D did not cross-link DNA template. These results suggest that transcription arrest is facilitated by electrostatic interactions between positively charged Nun residues Lys(106) and Lys(107) and negatively charged DNA phosphate groups. These may assist intercalation of Trp(108) into template.

Suppression of factor-dependent transcription termination by antiterminator RNA.

Nascent transcripts of the phage HK022 put sites modify the transcription elongation complex so that it terminates less efficiently at intrinsic transcription terminators and accelerates through pause sites. We show here that the modification also suppresses termination in vivo at two factor-dependent terminators, one that depends on the bacterial Rho protein and a second that depends on the HK022-encoded Nun protein. Suppression was efficient when the termination factors were present at physiological levels, but an increase in the intracellular concentration of Nun increased termination both in the presence and absence of put. put-mediated antitermination thus shows no apparent terminator specificity, suggesting that put inhibits a step that is common to termination at the different types of terminator.

Solution structure and stability of the full-length excisionase from bacteriophage HK022.

Heteronuclear high-resolution NMR spectroscopy was employed to determine the solution structure of the excisionase protein (Xis) from the lambda-like bacteriophage HK022 and to study its sequence-specific DNA interaction. As wild-type Xis was previously characterized as a generally unstable protein, a biologically active HK022 Xis mutant with a single amino acid substitution Cys28-->Ser was used in this work. This substitution has been shown to diminish the irreversibility of Xis denaturation and subsequent degradation, but does not affect the structural or thermodynamic properties of the protein, as evidenced by NMR and differential scanning calorimetry. The solution structure of HK022 Xis forms a compact, highly ordered protein core with two well-defined alpha-helices (residues 5-11 and 18-27) and five beta-strands (residues 2-4, 30-31, 35-36, 41-44 and 48-49). These data correlate well with 1H2O-2H2O exchange experiments and imply a different organization of the HK022 Xis secondary structure elements in comparison with the previously determined structure of the bacteriophage lambda excisionase. Superposition of both Xis structures indicates a better correspondence of the full-length HK022 Xis to the typical 'winged-helix' DNA-binding motif, as found, for example, in the DNA-binding domain of the Mu-phage repressor. Residues 51-72, which were not resolved in the lambda Xis, do not show any regular structure in HK022 Xis and thus appear to be completely disordered in solution. The resonance assignments have shown, however, that an unusual connectivity exists between residues Asn66 and Gly67 owing to asparagine-isoaspartyl isomerization. Such an isomerization has been previously observed and characterized only in eukaryotic proteins.