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Capture of a Recombination Activating Sequence from Mammalian Cells

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Capture of a Recombination Activating Sequence from Mammalian Cells Gene Therapy (1999) 6, 1819–1825 1999 Stockton Press All rights reserved 0969-7128/99 $15.00 http://www.stockton-press.co.uk/gt Capture of a recombination activating sequence from mammalian cells P Olson and R Dornburg The Dorrance H Hamilton Laboratories, Center for Human Virology, Division of Infectious Diseases, Jefferson Medical College, Thomas Jefferson University, Jefferson Alumni Hall, 1020 Locust Street, Rm 329, Philadelphia, PA 19107, USA We have developed a genetic trap for identifying revealed a putative recombination signal (CCCACCC). sequences that promote homologous DNA recombination. When this heptamer or an abbreviated form (CCCACC) The trap employs a retroviral vector that normally disables were reinserted into the vector, they stimulated vector itself after one round of replication. Insertion of defined repair and other DNA rearrangements. Mutant forms of DNA sequences into the vector induced the repair of a 300 these oligomers (eg CCCAACC or CCWACWS) did not. base pair deletion, which restored its ability to replicate. Our data suggest that the recombination events occurred Tests of random sequence libraries made in the vector within 48 h after transfection. Keywords: recombination; retroviral vector; vector stability; gene conversion; gene therapy Introduction scripts are still made from the intact left LTR, but reverse transcription copies the deletion to both LTRs, disabling Recognition of cis-acting DNA signals occupies a central the daughter provirus.15–17 We found that the SIN vectors role in both site-specific and general recombination path- that could escape this programmed disablement did so 1–6 ways. Signals in site-specific pathways define the by recombinationally repairing the U3-deleted LTR. DNA points at which the exchange will occur. General signals sequence analysis of reconstituted LTRs indicated that do not promote defined rearrangements but rather create the U3 repair was error-prone, and occasionally resulted hotspots by increasing the frequency of homologous in only partial repair of the deletion. LTR sequences in recombination in their vicinity. The best studied general the helper cell genome and in the left LTR served as tem- signal is E. coli’s crossover hotspot instigator (chi), which plate to reconstitute the LTR deletion.13 Remarkably, such 7–12 functions at the inception of the recombination. Chi recombinogenic SINs were constructed directly from acts as a biochemical switch for the RecBCD exonuclease– stable precursor vectors by sequence insertion. The two helicase complex. Beginning at a double-strand break, the stimulatory sequences studied were the neomycin resist- RecBCD complex processes exonucleolytically inward ance gene and the murine leukemia virus (MLV) encap- from the lesion site until it encounters a chi site. Chi cata- sidation sequence, psi (χ).18,19 In this study, we tested ran- lyses the release of the RecD subunit, attenuating the dom sequences for their ability to promote such complex’s exonuclease activity without affecting its hel- recombination. These investigations led to the identifi- icase activity. The region unwound after chi is coated by cation of a heptamer (CCCACCC), which is frequently RecA monomers during invading strand preparation. found near hotspots of mammalian recombination. Although hotspots exist in eukaryotes, specific signals, mostly nuclease sites, have only been demonstrated in yeast. The recombination hotspots observed in higher Results eukaryotes typically lie near regions of highly repetitive sequences and are generally observed in the context of Experimental system 3–5 meiosis. The existence of ubiquitous, general somatic To analyze the unusually high frequency of retroviral 5 recombination signals has been proposed, but nothing vector reconstitution and to identify specific sequences comparable to chi has yet been functionally identified. that trigger this process, we developed a retroviral vector We had previously seen evidence for general recombi- derived ‘recombination activator trap’ (Figure 1). This nation signals while investigating a group of self-inactiv- trap employs the retroviral vector pRD17 which self-inac- ating (SIN) retroviral vectors that were able to escape tivates after one round of retroviral replication as 13,14 their programmed inactivation efficiently. Normally, expected and the retroviral packaging line C3A2.20–22 SIN vectors are restricted to one round of replication by C3A2 cells contain multiple copies of the SNV LTR to a deletion that removes U3 from the provirus’s right long express retroviral Gag-pol and Env proteins. The pRD17 terminal repeat (LTR, Figure 1). Vector genomic tran- vector contains a selectable marker gene, which confers resistance to hygromycin B and has been used exten- sively to study cDNA gene formation by retroviruses.20 Correspondence: R Dornburg This vector has been shown to be completely stable and Received 1 February 1999; accepted 15 July 1999 no vector rearrangements have been observed. By Recombination activating sequences P Olson and R Dornburg 1820 Figure 1 Recombination activator trap. A retroviral vector system has been used to isolate recombination activating DNA sequences from random sequences cloned into a self-inactivating (SIN) retroviral vector (pRD17, top). pRD17 contains the hygromycin B phosphotransferase gene under the control of an internal promoter, allowing hygromycin selection of transfected or infected cells. In Step 1, plasmid DNAs containing pRD17 SIN vectors are transfected into the retroviral packaging line C3A2, which contains multiple copies of the SNV-LTR. Virus is harvested from such cells and fresh C3A2 helper cells are infected (Step 2). If recombination (triggered by a specific DNA recombination activator, RA) in transfected Step 1 cells reconstitutes the deletion in the right LTR, a provirus with complete LTRs will be formed in Step 2 helper cells. RNA transcribed from this provirus can be packaged and transduced to fresh target cells (Step 3). Vectors without a recombination activator will copy the deleted right LTR to both ends of the provirus and, hence, will replicate only once. Thus, selection of cells infected with virus harvested from Step 2 helper cells, can lead to the isolation of vector proviruses containing a recombination activating sequence. This RA can be recovered by PCR (Figure 2). creating DNA libraries in the vector, we now use it as a jected to the experimental protocol (Figure 1), about 200 trap for identifying recombination signals (Figure 1, top). hygromycin-resistant colonies were detected after the The insertion of a specific recombination activator (RA) second round of retroviral replication. When a control was expected to trigger recombination leading to a vector with two full-length LTRs was subjected to this repaired vector LTR using LTR templates in the C3A2 experimental protocol, about 106 hygromycin-resistant helper cell. Thus, subjecting such vectors to two rounds colonies were observed at step 3 (data not shown). These of retroviral replication enabled the identification of vec- data suggest that only a rather small number of the ran- tor species that underwent recombination (Figure 1), dom inserts was able to trigger vector repair. Further- because vectors without RAs replicate only once. more, a smaller library containing about 500 random A retroviral vector library was constructed by inserting sequences failed to produce any retroviral vectors with size-selected (400 bases or smaller) Sau3Al-digested D17 reconstituted LTRs. Latter data indicate that the small chromosomal DNA into the BamHI site of pRD17 library did not carry recombinogenic DNA sequences. (Figure 1). The restriction enzymes Sau3Al and BamHl To determine whether vectors with reconstituted LTRs create identical 39 overhangs after cutting the target had an insert with a common sequence motif, the DNA. Thus, they can be easily ligated with each other.23 chromosomal DNAs of hygromycin-resistant colonies After transformation of the ligation products into E. coli, were isolated. Inserts from vectors with reconstituted the number and size of inserts was determined by analyz- LTRs were recovered by PCR amplification using primers ing plasmid DNAs recovered from 20 bacterial clones. specific for flanking vector sequences (Figure 2). PCR The rest of the bacterial colonies which contained more products were cloned and sequenced (Figure 2 and data than 5000 unique inserts were pooled and plasmid DNAs not shown). were isolated. When this plasmid vector library was sub- Surprisingly, three vectors’ DNAs (recovered from Recombination activating sequences P Olson and R Dornburg 1821 Figure 2 Identification of recombination activators. The retroviral vector pRD17 is shown at the top. Inserts from proviruses with reconstituted LTRs were recovered PCR amplification using primers (indicated by the arrows) specific for regions flanking the BamHI cloning site. The sequence at the top is the original plasmid sequence of pRD17. The sequences below (pRA2, pRA4 and pRA9) were recovered from DNAs isolated from Step 3 cell clones. A dot indicates sequence homology; a dash indicates a missing bp. The box indicates the inserts found in clones pRA2, pRA4 and pRA9. three separate transfections) contained only a short four nucleotide insert (CCCA) which created a symmetric sequence element, CCCACCC (Figure 2). In each case, the Table 1 Reinsertion of the CCCACCC motif into pRD17 stimu- lates LTR repair clone had apparently undergone recombination, which deleted the remainder of the insert
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