Wild-type Int Research Articles

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.

Modulation of the Vault Protein-Protein Interaction for Tuning of Molecular Release

Vaults are naturally occurring ovoid nanoparticles constructed from a protein shell that is composed of multiple copies of major vault protein (MVP). The vault-interacting domain of vault poly(ADP-ribose)-polymerase (INT) has been used as a shuttle to pack biomolecular cargo in the vault lumen. However, the interaction between INT and MVP is poorly understood. It is hypothesized that the release rate of biomolecular cargo from the vault lumen is related to the interaction between MVP and INT. To tune the release of molecular cargos from the vault nanoparticles, we determined the interactions between the isolated INT-interacting MVP domains (iMVP) and wild-type INT and compared them to two structurally modified INT: 15-amino acid deletion at the C terminus (INTΔC15) and histidine substituted at the interaction surface (INT/DSA/3 H) to impart a pH-sensitive response. The apparent affinity constants determined using surface plasmon resonance (SPR) biosensor technology are 262 ± 4 nM for iMVP/INT, 1800 ± 160 nM for iMVP/INTΔC15 at pH 7.4. The INT/DSA/3 H exhibits stronger affinity to iMVP (KDapp = 24 nM) and dissociates at a slower rate than wild-type INT at pH 6.0.

Receipt of the C-terminal Tail from a Neighboring λ Int Protomer Allosterically Stimulates Holliday Junction Resolution

Bacteriophage lambda integrase (Int) catalyzes the integration and excision of the phage lambda chromosome into and out of the Esherichia coli host chromosome. The seven carboxy-terminal residues (C-terminal tail) of Int comprise a context-sensitive regulatory element that links catalytic function with protein multimerization and also coordinates Int functions within the multimeric recombinogenic complex. The experiments reported here show that the beta5-strand of Int is not simply a placeholder for the C-terminal tail but rather exerts its own allosteric effects on Int function in response to the incoming tail. Using a mutant integrase in which the C-terminal tail has been deleted (W350ter), we demonstrate that the C-terminal tail is required for efficient and accurate resolution of Holliday junctions by tetrameric Int. Addition of a free heptameric peptide of the same sequence as the C-terminal tail partially reverses the W350ter defects by stimulating Holliday junction resolution. The peptide also stimulates the topoisomerase function of monomeric W350ter. Single residue alterations in the peptide sequence and a mutant of the beta5 strand indicate that the observed stimulation arises from specific contacts with the beta5 strand (residues 239-243). The peptide does not stimulate binding of W350ter to its cognate DNA sites and therefore appears to recapitulate the effects of the normal C-terminal tail intermolecular contacts in wild-type Int. Models for the allosteric stimulation of Int activity by beta5 strand contacts are discussed.

The Efficiency of Mispaired Ligations by λ Integrase is Extremely Sensitive to Context

The integrase protein (Int) of phage lambda is a well-studied representative of the tyrosine recombinase family, whose defining features are two sequential pairs of DNA cleavage/ligation reactions that proceed via a 3' phosphotyrosine covalent intermediate to first form and then resolve a Holliday junction recombination intermediate. We devised an assay that takes advantage of DNA hairpin formation at one Int target site to trap Int cleavages at a different target site, and thereby reveal iterative cycles of cleavage and ligation that would otherwise be undetected. Using this assay and others to compare wild-type Int and a mutant (R169D) defective in forming proper dimer/tetramer interfaces, we found that the efficiency of "bottom-strand" DNA cleavage by wild-type Int, but not R169D, is very sensitive to the base-pair at the "top-strand" cleavage site, seven base-pairs away. We show that this is related to the finding that hairpin formation involving ligation of a mispaired base is much faster for R169D than for wild-type Int, but only in the context of a multimeric complex. During resolution of Holliday junction recombination intermediates, wild-type Int, but not R169D, is very sensitive to homology at the sites of ligation. A long-sought insight from these results is that during Holliday junction resolution the tetrameric Int complex remains intact until after ligation of the product helices has been completed. This contrasts with models in which the second pair of DNA cleavages is a trigger for dissolution of the recombination complex.

Two structural features of lambda integrase that are critical for DNA cleavage by multimers but not by monomers.

Despite many years of genetic and biochemical studies on the lambda integrase (Int) recombination system, it is still not known whether the Int protein is competent for DNA cleavage as a monomer. We have addressed this question, as part of a larger study of Int functions critical for the formation of higher-order complexes, by isolating "multimer-specific" mutants. We identify a pair of oppositely charged residues, E153 and R169, that comprise an intermolecular salt bridge within a functional Int multimer. Mutation of either of these residues significantly reduces both the cleavage of full-att sites and the resolution of Holliday junctions without compromising the cleavage of half-att site substrates. Allele-specific suppressor mutations were generated at these residues. Their interaction with wild-type Int on preformed Holliday junctions indicates that the mutated residues comprise an intermolecular salt bridge. We have also shown that the most C-terminal seven residues of Int, which comprise another previously identified subunit interface, inhibit DNA cleavage by monomeric but not multimeric Int. Taken together, our results lead us to conclude that Int can cleave DNA as a monomer. We also identify and discuss unique structural features of Int that act negatively to reduce its activity as a monomer and other features that act positively to enhance its activity as a multimer.

Attenuating Functions of the C Terminus of λ Integrase

The tyrosine family site-specific recombinases, in contrast to the related type I topoisomerases, which act as monomers on a single DNA molecule, rely on multi-protein complexes to synapse partner DNAs and coordinate two sequential strand exchanges involving four nicking–closing reactions. Here, we analyze three mutants of the catalytic domain of λ integrase (Int), A241V, I353M and W350ter that are defective for normal recombination, but possess increased topoisomerase activity. The mutant enzymes can carry out individual DNA strand exchanges using truncated substrates or Holliday junctions, and they show more DNA-cleavage activity than wild-type Int on isolated att sites. Structural modeling predicts that the substituted residues may destabilize interactions between the C-terminal β-strand (β7) of Int and the core of the protein. The cleavage-competent state of Int requires the repositioning of the nucleophile (Y342) located on β6 and the catalyst K235 located on the flexible β2-β3 loop, relative to their positions in a crystal structure of the inactive conformation. We propose that the anchoring of β7 against the protein core restrains the movement of Tyr342 and/or Lys235, causing an attenuation of cleavage activity in most contexts. Within a bona fide recombination complex, the release of strand β7 would allow Tyr342 and Lys235 to assume catalytically active conformations in coordination with other Int protomers in the complex. The loss of β7 packing by misalignment or truncation in the mutant proteins described here causes a loss of regulated activity, thereby favoring DNA cleavage activity in monomeric complexes and forfeiting the coordination of strand-exchange necessary for efficient recombination.

Site-specific recombination in human cells catalyzed by phage λ integrase mutants

Phage lambda Integrase (Int) is the prototype of the so-called integrase family of conservative site-specific recombinases, which includes Cre and FLP. The natural function of Int is to execute integration and excision of the phage into and out of the Escherichia coli genome, respectively. In contrast to Cre and FLP, however, wild-type Int requires accessory proteins and DNA supercoiling of target sites to catalyze recombination. Here, we show that two mutant Int proteins, Int-h (E174 K) and its derivative Int-h/218 (E174 K/E218 K), which do not require accessory factors, are proficient to perform intramolecular integrative and excisive recombination in co-transfection assays inside human cells. Intramolecular integrative recombination is also detectable by Southern analysis in human reporter cell lines harboring target sites attB and attP as stable genomic sequences. Recombination by wild-type Int, however, is not detectable by this method. The latter result implies that eukaryotic co-factors, which could functionally replace the prokaryotic ones normally required for wild-type Int, are most likely not present in human cells.

Characterization of the binding sites of two proteins involved in the bacteriophage P2 site-specific recombination system

Integration of the bacteriophage P2 genome into the Escherichia coli host chromosome occurs by site-specific recombination between the phage attP and E. coli attB sites. The phage-encoded 38-kDa protein, integrase, is known to be necessary for both phage integration as well as excision. In order to begin the molecular characterization of this recombination event, we have cloned the int gene and overproduced and partially purified the Int protein and an N-terminal truncated form of Int. Both the wild-type Int protein and the integration host factor (IHF) of E. coli were required to mediate integrative recombination in vitro between a supercoiled attP plasmid and a linear attB substrate. Footprint experiments revealed one Int-protected region on both of the attP arms, each containing direct repeats of the consensus sequence TGTGGACA. The common core sequences at attP and attB were also protected by Int from nuclease digestion, and these contained a different consensus sequence, AA T/A T/A C/A T/G CCC, arranged as inverted repeats at each core. A single IHF-protected site was located on the P (left) arm, placed between the core- and P arm-binding site for Int. Cooperative binding by Int and IHF to the attP region was demonstrated with band-shift assays and footprinting studies. Our data support the existence of two DNA-binding domains on Int, having unrelated sequence specificities. We propose that P2 Int, IHF, attP, and attB assemble in a higher-order complex, or intasome, prior to site-specific integrative recombination analogous to that formed during lambda integration.

Self-Limiting Infection by int/nef-Double Mutants of Simian Immunodeficiency Virus

Simian immunodeficiency virus (SlVmac) infectious for rhesus monkeys was altered by site-directed mutagenesis of genes influencing in vivo replication and persistence with the long-term goal to develop attenuated lentiviruses with limited replication capacity in vivo. Double mutants of SIVmac (termed delta-int 1 to 3) were generated by introducing frameshift and deletion mutations into the nef gene and into the pol gene region coding for the integrase protein. Delta-int/delta-nef viruses formed after transfection of CD4+-lymphocyte cell lines were unable to establish sustained replication. In contrast, both wild-type SIVmac and mutant SIVmac delta-nef (coding for a truncated NEF protein and a wild-type INT protein) replicated continuously and at a comparable rate. However, a transient and self-limiting infection of the C8166 T-cell line was observed subsequent to transfection of double mutant proviruses into HeLa-tat-III cells. Viruses attenuated by int/nef-double mutation were able to enter the T-cells, initiate synthesis of viral DNA as shown by PCR amplification of closed circular episomes, and express viral antigens in infected cells as demonstrated by immunocytochemical staining. Integration of the int mutant viruses into the chromosome was completely inhibited. Episomal viral DNA was detectable in the infected cells for up to 2 weeks, after which it disappeared. Thus, SIVmac attenuated by int and nef mutation established a transient infection of permissive cells resulting in the expression of viral antigen from episomal viral DNA over a limited period of time.

Conjugative transposition of Tn916: the transposon int gene is required only in the donor.

Conjugative transposition of transposon Tn916 has been shown to proceed by excision of the transposon in the donor strain and insertion of this element in the recipient. This process requires the product of the transposon int gene. We report here the surprising finding that the int gene is required only in the donor during conjugative transposition. We find that Tn916 int-1, whose int gene has been inactivated by an insertion mutation, transposes when a complementing wild-type int gene is present only in the donor during mating. When the int+ gene is present in a plasmid and is expressed from the spac promoter, conjugative transposition is very inefficient. However, when the Int+ function is supplied from a coresident distantly linked Tn916 tra-641 mutant, which is defective in a function required for conjugation, efficient conjugative transposition of Tn916 int-1 occurs. This suggests either that Int is not required for integration of Tn916 in gram-positive bacteria or that the protein is transferred from the donor to the transconjugant during the mating event. When the nonconjugative plasmid pAT145 was present in the donor, it was rarely cotransferred with Tn916. This suggests that complete fusion of mating cells is not common during conjugative transposition.

Mutational analysis of the lambda int gene: DNA sequence of dominant mutations.

We have combined techniques of genetic and physical mapping with rapid DNA sequence analysis to identify the nucleotide change in lambda int mutations. These mutations define two dominant phenotypic classes: (i) recombination that is partially independent of accessory factors, and (ii) inhibition of wild-type Int by missense or nonsense proteins, i.e., negative complementation.

Purification and properties of Int-h, a variant protein involved in site-specific recombination of bacteriophage lambda.

Under physiological conditions, integration of lambda DNA into the Escherichia coli chromosome requires the direct participation of only two proteins, the viral int gene product and E. coli integration host factor (IHF). A variant of the int gene has been isolated that permits integrative recombination in cells mutant for one of the two subunits of IHF (Miller, H.I., Mozola, M.A., and Friedman, D.I. (1980) Cell 20, 721-729). In the present work, we have purified Int-h, the product of this variant gene. In contrast to the wild-type int gene product (Int+), which produces almost no recombinants in the absence of IHF, purified Int-h protein sponsors reduced but significant levels of integrative recombination in the absence of any E. coli supplement. This shows that the int gene encodes all the information necessary for the elementary steps in recombination and implies that IHF functions as an accessory protein. When supplemented by IHF, recombination promoted by Int-h resembles that promoted by Int+ in kinetics, stoichiometry of Int and IHF, and nature of the recombinant product. Under these conditions, Int-h uses supercoiled DNA more effectively than nonsupercoiled DNA as a substrate for recombination, as does Int+. However, in the absence of IHF, Int-h recombines supercoiled and nonsupercoiled substrates identically, indicating that IHF is an important part of the mechanism that senses the supercoiled state of the substrate DNA during recombination. A surprising difference in recombination carried out by Int-h in the presence or absence of IHF concerns the degree to which sites on the same circle recombine with one another as opposed to sites on sister molecules. In the presence of IHF, Int-h favors intramolecular recombination, as does Int+. However, in the absence of IHF, Int-h almost exclusively promotes intermolecular recombination.

Retroregulation of the int gene of bacteriophage lambda: control of translation completion.

Bacteriophage lambda regulates the integration--excision reaction as a crucial aspect of the choice of pathway during lysogenic or lytic viral development. This control involves differential expression of the tightly linked, partially overlapping int and xis genes from two promoter sites: pI, positively regulated by cII/cIII proteins, and pL, positively regulated by N protein. After lambda infection, Int is synthesized from the pI transcript under cII regulation; however, very little Int is produced from the pL RNA because of the existence of a cis-acting regulatory element, sib, on the opposite side of the int gene from the pL promoter. Presumably sib serves to prevent unwanted synthesis of Int protein during the lytic response; the Int protein necessary for excisive recombination from a prophage can be supplied by pL transcription because sib is separated from int by prophage insertion. We have studied the effect of sib on nearby lambda genes by means of gel electrophoresis of labeled proteins from infected cells. Deletion of the sib region greatly enhances production of Int protein without substantial effect on Xis production; thus, sib regulation normally is highly specific for Int. When the sib region is moved past int and xis by deletion, regulation of the adjacent gene for the protein Ea22 occurs, suggesting that sib regulation can work on other genes. Although synthesis of wild-type Int is severely inhibited by sib, shorter Int protein fragments generated by nonsense mutations escape sib regulation, indicating that the regulation is translational and occurs near the completion stage of protein synthesis. Regulation by sib thus exhibits novel regulatory features: distal location, recombinational control, and regulation of the completion of protein synthesis. Because Int and Ea22 control is lost in a RNase III- host, we suggest that sib regulation might involve RNase III cleavage of a RNA duplex region that includes sib and the regulatory target (normally the int gene). We note such a potential site within int.