Factors required in vitro for excision of the Bacteroides conjugative transposon, CTnDOT

Four genes have been found to be essential for excision of the Bacteroides conjugative transposon CTnDOT in vivo: intDOT, orf2c, orf2d, and exc. The intDOT gene encodes an integrase that is essential for integration and excision. The function of the other genes is still uncertain. Previously, we developed an in vitro system for the integration reaction. We have now developed an in vitro system for excision. In this system, the left and right junctions of CTnDOT, attL, and attR, are provided on separate plasmids. The excision reaction produced a cointegrate which contained the attDOT (the joined ends of CTnDOT) and attB (the chromosomal target site). Cointegrate formation was observed after electroporation of Escherichia coli with the assay mixture and was also detected directly in the assay mixture by Southern hybridization. The highest reaction frequencies (10(-3)) were obtained with a mixture that contained purified IntDOT and a cell extract from Bacteroides thetaiotaomicron 4001, which contained the excision region of CTnDOT carried on a plasmid. An unexpected finding was that the addition of purified Exc, which is essential for excision in vivo, was not required for excision in vitro, nor did it increase the frequency of cointegrate formation.     

Integration and excision of a Bacteroides conjugative transposon, CTnDOT

Bacteroides conjugative transposons (CTns) are thought to transfer by first excising themselves from the chromosome to form a nonreplicating circle, which is then transferred by conjugation to a recipient. Earlier studies showed that transfer of most Bacteroides CTns is stimulated by tetracycline, but it was not known which step in transfer is regulated. We have cloned and sequenced both ends of the Bacteroides CTn, CTnDOT, and have used this information to examine excision and integration events. A segment of DNA that contains the joined ends of CTnDOT and an adjacent open reading frame (ORF), intDOT, was necessary and sufficient for integration into the Bacteroides chromosome. Integration of this miniature form of the CTn was not regulated by tetracycline. Excision of CTnDOT and formation of the circular intermediate were detected by PCR, using primers designed from the end sequences. Sequence analysis of the PCR products revealed that excision and integration involve a 5-bp coupling sequence-type mechanism possibly similar to that used by CTn Tn916, a CTn found originally in enterococci. PCR analysis also demonstrated that excision is a tetracycline-regulated step in transfer. The integrated minielement containing intDOT and the ends of CTnDOT did not excise, nor did a larger minielement that also contained an ORF located immediately downstream of intDOT designated orf2. Thus, excision involves other genes besides intDOT and orf2. Both intDOT and orf2 were disrupted by single-crossover insertions. Analysis of the disruption mutants showed that intDOT was essential for excision but orf2 was not. Despite its proximity to the integrase gene, orf2 appears not to be essential for excision.     

The Bacteroides mobilizable insertion element, NBU1, integrates into the 3' end of a Leu-tRNA gene and has an integrase that is a member of the lambda integrase family.

NBU1 is a 10.3-kbp integrated Bacteroides element that can be induced to excise from the chromosome and can be mobilized to a recipient by trans-acting functions provided by certain Bacteroides conjugative transposons. The NBU1 transfer intermediate is a covalently closed circle, which is presumed to be the form that integrates into the recipient genome. We report here that a 2.4-kbp segment of NBU1 was all that was required for site-specific integration into the chromosome of Bacteroides thetaiotaomicron 5482. This 2.4-kbp region included the joined ends of the NBU1 circular form (attN1) and a single open reading frame, intN1, which encoded the integrase. Previously, we had found that NBU1 integrates preferentially into a single site in B. thetaiotaomicron 5482. We have now shown that the NBU1 target site is located at the 3' end of a Leu-tRNA gene. The NBU1 integrase gene, intN1, was sequenced. The predicted protein had little overall amino acid sequence similarity to any proteins in the databases but had limited carboxy-terminal similarity to the integrases of lambdoid phages and to the integrases of the gram-positive conjugative transposons Tn916 and Tn1545. We also report that the intN1 gene is expressed constitutively.     

Identification of genes required for excision of CTnDOT, a Bacteroides conjugative transposon

Integrated self-transmissible elements called conjugative transposons have been found in many different bacteria, but little is known about how they excise from the chromosome to form the circular intermediate, which is then transferred by conjugation. We have now identified a gene, exc, which is required for the excision of the Bacteroides conjugative transposon, CTnDOT. The int gene of CTnDOT is a member of the lambda integrase family of recombinases, a family that also contains the integrase of the Gram-positive conjugative transposon Tn916. The exc gene was located 15 kbp from the int gene, which is located at one end of the 65 kbp element. The exc gene, together with the regulatory genes, rteA, rteB and rteC, were necessary to excise a miniature form of CTnDOT that contained only the ends of the element and the int gene. Another open reading frame (ORF) in the same operon and upstream of exc, orf3, was not essential for excision and had no significant amino acid sequence similarity to any proteins in the databases. The deduced amino acid sequence of the CTnDOT Exc protein has significant similarity to topoisomerases. A small ORF (orf2) that could encode a small, basic protein comparable with lambda and Tn916 excision proteins (Xis) was located immediately downstream of the CTnDOT int gene. Although Xis proteins are required for excision of lambda and Tn916, orf2 had no effect on excision of the element. Excision of the CTnDOT mini-element was not affected by the site in which it was integrated, another difference from Tn916. Our results demonstrate that the Bacteroides CTnDOT excision system is tightly regulated and appears to be different from that of any other known integrated transmissible element, including those of some Bacteroides mobilizable transposons that are mobilized by CTnDOT.     

IntDOT interactions with core- and arm-type sites of the conjugative transposon CTnDOT

CTnDOT is a Bacteroides conjugative transposon (CTn) that has facilitated the spread of antibiotic resistances among bacteria in the human gut in recent years. Although the integrase encoded by CTnDOT (IntDOT) carries the C-terminal set of conserved amino acids that is characteristic of the tyrosine family of recombinases, the reaction it catalyzes involves a novel step that creates a short region of heterology at the joined ends of the element during recombination. Also, in contrast to tyrosine recombinases, IntDOT catalyzes a reaction that is not site specific. To determine what types of contacts IntDOT makes with the DNA during excision and integration, we first developed an agarose gel-based assay for CTnDOT recombination, which facilitated the purification of the native IntDOT protein. The partially purified IntDOT was then used for DNase I footprinting analysis of the integration site attDOT and the excision sites attL and attR. Our results indicate that CTnDOT has five or six arm sites that are likely to be involved in forming higher-order nucleoprotein complexes necessary for synapsis. In addition, there are four core sites that flank the sites of strand exchange during recombination. Thus, despite the fact that the reaction catalyzed by IntDOT appears to be different from that typically catalyzed by tyrosine recombinases, the protein-DNA interactions required for higher-order structures and recombination appear to be similar.     

Integration and excision of a newly discovered bacteroides conjugative transposon, CTnBST

Conjugative transposons (CTns) are major contributors to the spread of antibiotic resistance genes among Bacteroides species. CTnBST, a newly discovered Bacteroides conjugative transposon, carries an erythromycin resistance gene, ermB, and previously has been estimated to be about 100 kbp in size. We report here the locations and sequencing of both of its ends. We have also located and sequenced the gene that catalyzes the integration of CTnBST, intBST. The integrase gene encodes a 377-amino-acid protein that has the C-terminal R-K-H-R-H-Y motif that is characteristic of members of the tyrosine recombinase family of integrases. DNA sequence comparisons of the ends of CTnBST, the joined ends of the circular intermediate, and the preferred site into which the circular form of CTnBST had integrated revealed that the preferred integration site (attB1) contained an 18-bp sequence of identity to the crossover region, attBST, on CTnBST. Although this site was used in about one-half of the integration events, sequence analysis of these integration events revealed that both CTnBST and a miniature form of CTnBST (miniBST) integrated into a variety of other sites in the chromosome. All of the sites had two conserved regions, AATCTG and AAAT. These two regions flanked a 2-bp sequence, bp 10 and bp 11 of the 18-bp sequence, that varied in some of the different sites and sometimes in the attBST sequences. Our results suggest that CTnBST integrates site selectively and that the crossover appears to occur within a 12-bp region that contains the two regions of conserved sequences.     

NBU1 integrase: evidence for an altered recombination mechanism

NBU1 is a 10.3 kbp Bacteroides mobilizable transposon. A previous study had identified a 2.7 kbp segment of the excised circular intermediate that was sufficient to mediate integration of the element after transfer. This segment contained an integrase gene, intN1, and a region spanning the ends of the circular form within which integration occurred (attN1). The integrase protein, IntN1, appeared to be a member of the tyrosine recombinase family because it contains the canonical C-terminal RKHRHY [RK(H/K)R(H/W)Y] motif that characterizes members of that family. In this study, we describe an Escherichia coli-based integration assay system that has allowed us to characterize attN1 in detail. We first localized attN1 to a 250 bp region. We then used site-directed mutations to identify directly repeated sequences within attN1 that were required for site-specific integration. The locus of NBU1 site-specific integration in the Bacteroides thetaiotaomicron chromosome, attBT1-1, contains a 14 bp sequence that is identical to a 14 bp sequence that spans the joined ends of the NBU1 attN1 site (common core sequences). The effects of mutations in the common core were different from the expected results if NBU1 integration was similar to lambda integration. In particular single base changes near one end of the common core region, which introduced heterology, actually increased the frequency of integration. By contrast, compensating changes that restored homology in the common core region reduced the integration frequency. The recombination mechanism also differs from the one used by conjugative transposons that have coupling sequences between the sites of strand cleavage and exchange. These results indicate that although NBU1 integrase is considered to be a member of the tyrosine recombinase family, it catalyses an integrative recombination reaction that occurs by a different crossover mechanism.     

NBU1, a mobilizable site-specific integrated element from Bacteroides spp., can integrate nonspecifically in Escherichia coli.

NBU1 is an integrated Bacteroides element that can he mobilized from Bacteroides donors to Bacteroides recipients. Previous studies have shown that a plasmid carrying the internal mobilization region of NBU1 could be transferred by conjugation from Bacteroides thetaiotaomicron to Escherichia coli. In this report, we show that NBU1 can integrate in E. coli. Whereas integration of NBU1 in B. thetaiotaomicron is site specific, integration of NBU1 in E. coli was relatively random, and the insertion frequency of NBU1 into the E. coli chromosome was 100 to 1,000 times lower than the frequency of integration in B. thetaiotaomicron. The frequency of NBU1 integration in E. coli could be increased about 10- to 70-fold, to a value close to that seen with B. thetaiotaomicron, if the primary integration site from B. thetaiotaomicron, BT1-1, was provided on a plasmid in the E. coli recipient or the NBU1 integrase gene, intN1, was provided on a high-copy-number plasmid to increase the amount of integrase available in the recipient. When the primary integration site was available in the recipient, NBU1 integrated site specifically in E. coli. Our results show that NBUs have a very broad host range and are capable of moving from Bacteroides spp. to distantly related species such as E. coli. Moreover, sequence analysis of NBU1 integration sites provided by integration events in E. coli has helped to identify some regions of the NBU1 attachment site that may play a role in the integration process.     

Retroviral integration: in vitro host site selection by avian integrase.

Viral integrase catalyzes the integration of the linear viral DNA genome into the chromatin of the infected host cell, an essential step in the life cycle of retroviruses. The reaction produces a characteristic small duplication of host sequences at the site of integration, implying that there is a close juxtaposition of the viral DNA ends during a concerted integration event. We have used an in vitro assay to measure the concerted integration of virus-like plasmid DNA into naked lambda DNA catalyzed by virion purified avian integrase. In contrast to in vivo avian integration, which has strong fidelity for a 6-bp duplication, purified avian integrase in the context of this assay produced a distribution of duplication sizes, with the 6-bp size dominating. The metal cofactor Mg2+ induced increased fidelity for the 6-bp duplication relative to that with Mn2+. The immediate sequence of the host site may also influence duplication size in that we found sites that sustained multiple independent integration events producing the same duplication size. Additionally, for each set of cloned integration sites (5, 6, and 7 bp), a unique but similar symmetrical pattern of G/C and A/T sequence biases was found. Using duplex oligonucleotides as target substrates, we tested the significance of the 6-bp G/C and A/T pattern for site selection. In the context of this assay, which is likely dominated by the integration of only one viral end, the 6-bp pattern was not preferred. Instead, integration was predominantly into the 3' ends of the oligonucleotides. The combined results of the lambda and oligonucleotide assays indicated that although host site selection has properties in common with recognition of the viral DNA termini, the nonrandom sequence preferences seen for host site selection were not identical to the sequence requirements for long terminal repeat recognition.     

Multiple gene products and sequences required for excision of the mobilizable integrated Bacteroides element NBU1

NBU1 is an integrated 10.3-kbp Bacteroides element, which can excise and transfer to Bacteroides or Escherichia coli recipients, where it integrates into the recipient genome. NBU1 relies on large, >60-kbp, conjugative transposons for factors that trigger excision and for mobilization of the circular form to recipients. Previously, we showed that a single integrase gene, intN1, was necessary and sufficient for integration of NBU1 into its target site on the Bacteroides or E. coli genome. We now show that an unexpectedly large region of NBU1 is required for excision. This region includes, in addition to intN1, four open reading frames plus a large region downstream of the fourth gene, prmN1. This downstream sequence was designated XRS, for "excision-required sequence." XRS contains the oriT of the circular form of NBU1 and about two-thirds of the adjacent mobilization gene, mobN1. This is the first time an oriT, which is involved in conjugal transfer of the circular form, has been implicated in excision. Disruption of the gene immediately downstream of intN1, orf2, completely abolished excision. The next open reading frame, orf2x, was too small to be disrupted, so we still do not know whether it plays a role in the excision reaction. Deletions were made in each of two open reading frames downstream of orf2x, orf3 and prmN1. Both of these deletions abolished excision, indicating that these genes are also essential for excision. Attempts to complement various mutations in the excision region led us to realize that a portion of the excision region carrying prmN1 and part of the XRS (XRS(HIII)) inhibited excision when provided in trans on a multicopy plasmid (8 to 10 copies per cell). However, a fragment carrying prmN1, XRS, and the entire mobilization gene, mobN1, did not have this effect. The smaller fragment may be interfering with excision by attracting proteins made by the intact NBU1 and thus removing them from the excision complex. Our results show clearly that excision is a complex process that involves several proteins and a cis-acting region (XRS) which includes the oriT. We suggest that this complex excision machinery may be necessary to allow NBU1 to coordinate nicking at the ends during excision and nicking at the oriT during conjugal transfer, to prevent premature nicking at the oriT before NBU1 has excised and circularized.     

Juxtaposition of two viral DNA ends in a bimolecular disintegration reaction mediated by multimers of human immunodeficiency virus type 1 or murine leukemia virus integrase.

Integration of retroviral DNA involves a coordinated joining of the two ends of a viral DNA molecule into precisely spaced sites on target DNA. In this study, we designed an assay that requires two separate oligonucleotides to be brought together via interactions between integrase promoters to form a "crossbones" substrate that mimics the integration intermediate. The crossbones substrate contains two viral DNA ends, each joined to one strand of target DNA and separated by a defined length of target DNA. We showed that purified integrases of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV) could mediate a concerted strand cleavage-ligation between the two half-substrates at one or both viral DNA joining sites (trans disintegration). Another major product, termed fold-back, resulted from an intramolecular attack on the phosphodiester bond at the viral-target DNA junction by the 3'-OH group of the same DNA molecule (cis disintegration). The activity of integrase on the crossbones substrate depended on the presence of viral DNA sequences. For trans disintegration, the optimal length of target DNA between the viral DNA joining sites of the crossbones substrate corresponded to the spacing between the staggered joints formed on two opposite strands of target DNA during retroviral DNA integration in vivo. The activity of integrases on crossbones did not require complementary base pairing between the two half-substrates, indicating that the half-substrates were juxtaposed solely through protein-DNA interactions. The crossbones assay, therefore, measures the ability of integrase to juxtapose two viral DNA ends, an activity which heretofore has been difficult to detect by using purified integrase in conventional assays. Certain mutant integrases that were otherwise inactive with the crossbones substrate could complement one another, indicating that no single protomer in the integrase multimer requires a complete set of functional domains either for catalytic activity or for juxtaposition of the two viral DNA ends by the active multimer.     

Ty1 in vitro integration: effects of mutations in cis and in trans.

Retrotransposon Ty1 of Saccharomyces cerevisiae inserts a double-stranded Ty1 cDNA into the yeast genome by a reaction analogous to the integration mechanism used by retroviruses. A quantitative in vitro integration assay that directly detects integrative recombination products was developed for Ty1. Blunt-ended artificial radioactive substrates bearing Ty1 termini integrate into circular or linear target DNAs. The reaction is specific for native integrase isolated in the form of virus-like particles; virus-like particles prepared from integrase mutants were completely inactive in this assay. The products are radioactive, allowing direct detection after gel electrophoresis by autoradiography. Using this simple and amenable system, we characterized the biochemical requirements of the system and the structures of the major integration products. Two classes of products were detected: those that were the result of bona fide complete integration events (concerted reactions) and single-end joinings of substrate to target (half-reactions). Additionally, we used a genetic selection scheme to identify and characterize target sites of complete integration events into a circular target plasmid; a 5-bp target site duplication flanking the inserted DNA resembling the duplication characteristic of in vivo integration was observed.     

In vitro integration of retrotransposon Ty1: a direct physical assay.

Retrotransposon Ty1 of Saccharomyces cerevisiae inserts a double-stranded Ty1 cDNA into the yeast genome by a reaction analogous to the integration mechanism used by retroviruses. A quantitative in vitro integration assay that directly detects integrative recombination products was developed for Ty1. Blunt-ended artificial radioactive substrates bearing Ty1 termini integrate into circular or linear target DNAs. The reaction is specific for native integrase isolated in the form of virus-like particles; virus-like particles prepared from integrase mutants were completely inactive in this assay. The products are radioactive, allowing direct detection after gel electrophoresis by autoradiography. Using this simple and amenable system, we characterized the biochemical requirements of the system and the structures of the major integration products. Two classes of products were detected: those that were the result of bona fide complete integration events (concerted reactions) and single-end joinings of substrate to target (half-reactions). Additionally, we used a genetic selection scheme to identify and characterize target sites of complete integration events into a circular target plasmid; a 5-bp target site duplication flanking the inserted DNA resembling the duplication characteristic of in vivo integration was observed.     

Efficiency and Fidelity of Full-Site Integration Reactions Using Recombinant Simian Immunodeficiency Virus Integrase

Full-site integration by recombinant wild-type and mutant simian immunodeficiency virus (SIV) integrase (IN) was investigated with linear retrovirus-like DNA (469 bp) as a donor substrate and circular DNA (2,867 bp) as a target substrate. Under optimized conditions, recombinant SIV IN produced donor-target products consistent with full-site (two donor ends) and half-site (one donor end) reactions with equivalent frequency. Restriction enzyme analysis of the 3.8-kbp full-site reaction products confirmed the concerted insertion of two termini from separate donors into a single target molecule. Donor ends carrying the viral U5 termini were preferred over U3 termini for producing both half-site and full-site products. Bacterial genetic selection was used to isolate individual donor-target recombinants, and the donor-target junctions of the cloned products were characterized by sequencing. Analysis of 149 recombinants demonstrated approximately 84% fidelity for the appropriate simian retrovirus 5-bp host duplication. As seen previously in similar reactions with human immunodeficiency virus type 1 (HIV-1) IN from lysed virions, approximately 8% of the donor-target recombinants generated with recombinant SIV IN incurred specific 17- to 18- or 27- to 29-bp deletions. The efficiency and fidelity of the full-site integration reaction mediated by the purified, recombinant SIV IN is comparable to that of HIV-1 IN from virions. These observations suggest that a purified recombinant lentivirus IN is itself sufficient to recapitulate the full-site integration process.     

Circularized Ac/Ds Transposons: Formation, Structure and Fate

The maize Ac/Ds transposable elements are thought to transpose via a cut-and-paste mechanism, but the intermediates formed during transposition are still unknown. In this work we present evidence that circular Ac molecules are formed in plants containing actively transposing elements. In these circles, transposon ends are joined head-to-head. The sequence at the ends' junction is variable, containing small deletions or insertions. Circles containing deleted Ac ends are probably unable to successfully reintegrate. To test the ability of circles with intact transposon ends to integrate into the genome, an artificial Ds circle was constructed by cloning the joined ends of Ac into a plasmid carrying a plant selectable marker. When such a circular Ds was introduced into tobacco protoplasts in the presence of Ac-transposase, no efficient transposase-mediated integration was observed. Although a circular transposition intermediate cannot be ruled out, the findings of circles with deleted transposon ends and the absence of transposase-mediated integration of the circular Ds suggest that some of the joined-ends-carrying elements are not transposition intermediates, but rather abortive excision products. The formation of Ac circles might account for the previously described phenomenon of Ac-loss. The origin of Ac circles and the implications for models of Ac transposition are discussed.     

Retroviral DNA integration: reaction pathway and critical intermediates

The key DNA cutting and joining steps of retroviral DNA integration are carried out by the viral integrase protein. Structures of the individual domains of integrase have been determined, but their organization in the active complex with viral DNA is unknown. We show that HIV-1 integrase forms stable synaptic complexes in which a tetramer of integrase is stably associated with a pair of viral DNA ends. The viral DNA is processed within these complexes, which go on to capture the target DNA and integrate the viral DNA ends. The joining of the two viral DNA ends to target DNA occurs sequentially, with a stable intermediate complex in which only one DNA end is joined. The integration product also remains stably associated with integrase and likely requires disassembly before completion of the integration process by cellular enzymes. The results define the series of stable nucleoprotein complexes that mediate retroviral DNA integration.     

Integration site selection by the Bacteroides conjugative transposon CTnBST

A newly discovered Bacteroides conjugative transposon (CTn), CTnBST, integrates more site specifically than two other well-studied CTns, the Bacteroides CTn CTnDOT and the enterococcal CTn Tn916. Moreover, the integrase of CTnBST, IntBST, had the C-terminal 6-amino-acid signature that is associated with the catalytic regions of members of the tyrosine recombinase family, most of which integrate site specifically. Also, in most of these integrases, all of the conserved amino acids are required for integration. In the case of IntBST, however, we found that changing three of the six conserved amino acids in the signature, one of which was the presumed catalytic tyrosine, resulted in a 1,000-fold decrease in integration frequency. Changes in the other amino acids had little or no effect. Thus, although the CTnBST integrase still seems to be a member of the tyrosine recombinase family, it clearly differs to some extent from other members of the family in its catalytic site. We also determined the sequence requirements for CTnBST integration in the 18-bp region where the crossover occurs preferentially during integration. We found that CTnBST integrates in this preferred site about one-half of the time but can also use other sites. A consensus sequence was tentatively derived by comparison of a few secondary sites: AATCTGNNAAAT. We report here that within the consensus region, no single base change affected the frequency of integration. However, 3 bp at one end of the consensus sequence (CTG) proved to be essential for integration into the preferred site. This sequence appeared to be at one end of a 7-bp crossover region, CTGNNAA. The other bases could vary without affecting either integration frequency or specificity. Thus, in contrast to well-studied site-specific recombinases which require homology throughout the crossover region, integration of CTnBST requires homology at one end of the crossover region but not at the other end.     

In vitro HIV-1 selective integration into the target sequence and decoy-effect of the modified sequence

Although there have been a few reports that the HIV-1 genome can be selectively integrated into the genomic DNA of cultured host cell, the biochemistry of integration selectivity has not been fully understood. We modified the in vitro integration reaction protocol and developed a reaction system with higher efficiency. We used a substrate repeat, 5'-(GTCCCTTCCCAGT)(n)(ACTGGGAAGGGAC)(n)-3', and a modified sequence DNA ligated into a circular plasmid. CAGT and ACTG (shown in italics in the above sequence) in the repeat units originated from the HIV-1 proviral genome ends. Following the incubation of the HIV-1 genome end cDNA and recombinant integrase for the formation of the pre-integration (PI) complex, substrate DNA was reacted with this complex. It was confirmed that the integration selectively occurred in the middle segment of the repeat sequence. In addition, integration frequency and selectivity were positively correlated with repeat number n. On the other hand, both frequency and selectivity decreased markedly when using sequences with deletion of CAGT in the middle position of the original target sequence. Moreover, on incubation with the deleted DNAs and original sequence, the integration efficiency and selectivity for the original target sequence were significantly reduced, which indicated interference effects by the deleted sequence DNAs. Efficiency and selectivity were also found to vary discontinuously with changes in manganese dichloride concentration in the reaction buffer, probably due to its influence on the secondary structure of substrate DNA. Finally, integrase was found to form oligomers on the binding site and substrate DNA formed a loop-like structure. In conclusion, there is a considerable selectivity in HIV-integration into the specified sequence; however, similar DNA sequences can interfere with the integration process, and it is therefore difficult for in vivo integration to occur selectively in the actual host genome DNA.     

Efficient concerted integration of retrovirus-like DNA in vitro by avian myeloblastosis virus integrase.

We report the efficient concerted integration of a linear virus-like DNA donor into a 2.8 kbp circular DNA target by integrase (IN) purified from avian myeloblastosis virus. The donor was 528 bp, contained recessed 3' OH ends, was 5' end labeled, and had a unique restriction site not found in the target. Analysis of concerted (full-site) and half-site integration events was accomplished by restriction enzyme analysis and agarose gel electrophoresis. The donor also contained the SupF gene that was used for genetic selection of individual full-site recombinants to determine the host duplication size. Two different pathways, involving either one donor or two donor molecules, were used to produce full-site recombinants. About 90% of the full-site recombinants were the result of using two donor molecules per target. These results imply that juxtapositioning an end from each of two donors by IN was more efficient than the juxtapositioning of two ends of a single donor for the full-site reaction. The formation of preintegration complexes containing integrase and donor on ice prior to the addition of target enhanced the full-site reaction. After a 30 min reaction at 37 degrees C, approximately 20-25% of all donor/target recombinants were the result of concerted integration events. The efficient production of full-site recombinants required Mg2+; Mn2+ was only efficient for the production of half-site recombinants. We suggest that these preintegration complexes can be used to investigate the relationships between the 3' OH trimming and strand transfer reactions.     

Stereospecificity of reactions catalyzed by HIV-1 integrase

The retroviral integrase catalyzes two successive chemical reactions essential for integration of the retroviral genome into a host chromosome: 3' end processing, in which a dinucleotide is cleaved from each 3' end of the viral DNA; and the integration reaction itself, in which the resulting recessed 3' ends of the viral DNA are joined to the host DNA. We have examined the stereospecificity of human immunodeficiency virus type 1 integrase for phosphorothioate substrates in these reactions and in a third reaction, disintegration, which is macroscopically the reverse of integration. Integrase preferentially catalyzed end processing and integration of a substrate with the (R(p))-phosphorothioate stereoisomer at the reaction center and disintegration of a substrate with an (S(p))-phosphorothiate at the reaction center. These results suggest a model for the architecture of the active site of integrase, and its interactions with key features of the viral and target DNA.