Hirock J. Datta

The ability to stimulate recombination in a site-specific manner in mammalian cells may provide a useful tool for gene knockout and a valuable strategy for gene therapy. We previously demonstrated that psoralen adducts targeted by triple-helix-forming oligonucleotides (TFOs) could induce recombination between tandem repeats of a supF reporter gene in a simian virus 40 vector in monkey COS cells. Based on work showing that triple helices, even in the absence of associated psoralen adducts, are able to provoke DNA repair and cause mutations, we asked whether intermolecular triplexes could stimulate recombination. Here, we report that triple-helix formation itself is capable of promoting recombination and that this effect is dependent on a functional nucleotide excision repair (NER) pathway. Transfection of COS cells carrying the dual supF vector with a purine-rich TFO, AG30, designed to bind as a third strand to a region between the two mutant supF genes yielded recombinants at a frequency of 0.37%, fivefold above background, whereas a scrambled sequence control oligomer was ineffective. In human cells deficient in the NER factor XPA, the ability of AG30 to induce recombination was eliminated, but it was restored in a corrected subline expressing the XPA cDNA. In comparison, the ability of triplex-directed psoralen cross-links to induce recombination was only partially reduced in XPA-deficient cells, suggesting that NER is not the only pathway that can metabolize targeted psoralen photoadducts into recombinagenic intermediates. Interestingly, the triplex-induced recombination was unaffected in cells deficient in DNA mismatch repair, challenging our previous model of a heteroduplex intermediate and supporting a model based on end joining. This work demonstrates that oligonucleotide-mediated triplex formation can be recombinagenic, providing the basis for a potential strategy to direct genome modification by using high-affinity DNA binding ligands.

Triple helix-forming oligonucleotides (TFOs) can bind to polypurine/polypyrimidine regions in DNA in a sequence-specific manner. Triple helix formation has been shown to stimulate recombination in mammalian cells in both episomal and chromosomal targets containing direct repeat sequences. Bifunctional oligonucleotides consisting of a recombination donor domain tethered to a TFO domain were found to mediate site-specific recombination in an intracellular SV40 vector target. To elucidate the mechanism of triplex-induced recombination, we have examined the ability of intermolecular triplexes to provoke recombination within plasmid substrates in human cell-free extracts. An assay for reversion of a point mutation in the supFG1 gene in the plasmid pSupFG1/G144C was established in which recombination in the extracts was detected upon transformation into indicator bacteria. A bifunctional oligonucleotide containing a 30-nucleotide TFO domain linked to a 40-nucleotide donor domain was found to mediate gene correction in vitro at a frequency of 46 × 10−5, at least 20-fold above background and over 4-fold greater than the donor segment alone. Physical linkage of the TFO to the donor was unnecessary, as co-mixture of separate TFO and donor segments also yielded elevated gene correction frequencies. When the recombination and repair proteins HsRad51 and XPA were depleted from the extracts using specific antibodies, the triplex-induced recombination was diminished, but was either partially or completely restored upon supplementation with the purified HsRad51 or XPA proteins, respectively. These results establish that triplex-induced, intermolecular recombination between plasmid targets and short fragments of homologous DNA can be detected in human cell extracts and that this process is dependent on both XPA and HsRad51. Triple helix-forming oligonucleotides (TFOs) can bind to polypurine/polypyrimidine regions in DNA in a sequence-specific manner. Triple helix formation has been shown to stimulate recombination in mammalian cells in both episomal and chromosomal targets containing direct repeat sequences. Bifunctional oligonucleotides consisting of a recombination donor domain tethered to a TFO domain were found to mediate site-specific recombination in an intracellular SV40 vector target. To elucidate the mechanism of triplex-induced recombination, we have examined the ability of intermolecular triplexes to provoke recombination within plasmid substrates in human cell-free extracts. An assay for reversion of a point mutation in the supFG1 gene in the plasmid pSupFG1/G144C was established in which recombination in the extracts was detected upon transformation into indicator bacteria. A bifunctional oligonucleotide containing a 30-nucleotide TFO domain linked to a 40-nucleotide donor domain was found to mediate gene correction in vitro at a frequency of 46 × 10−5, at least 20-fold above background and over 4-fold greater than the donor segment alone. Physical linkage of the TFO to the donor was unnecessary, as co-mixture of separate TFO and donor segments also yielded elevated gene correction frequencies. When the recombination and repair proteins HsRad51 and XPA were depleted from the extracts using specific antibodies, the triplex-induced recombination was diminished, but was either partially or completely restored upon supplementation with the purified HsRad51 or XPA proteins, respectively. These results establish that triplex-induced, intermolecular recombination between plasmid targets and short fragments of homologous DNA can be detected in human cell extracts and that this process is dependent on both XPA and HsRad51. triple helix-forming oligonucleotide nucleotide excision repair nucleotide(s) base pair(s) dithiothreitol tethered donor high pressure liquid chromatography Targeted modification of the genome by gene replacement is of value as a research tool and has potential application to gene therapy. However, although facile methods exist to introduce new genes into mammalian cells, the frequency of homologous integration is limited (1Hanson K.D. Sedivy J.M. Mol. Cell. Biol. 1995; 15: 45-51Crossref PubMed Scopus (73) Google Scholar), and isolation of cells with site-specific gene insertion typically requires a selection procedure (2Capecchi M.R. Science. 1989; 244: 1288-1292Crossref PubMed Scopus (1634) Google Scholar). Site-specific DNA damage in the form of double-strand breaks produced by rare cutting endonucleases can promote homologous recombination at chromosomal loci in several cell systems (3Rouet P. Smih F. Jasin M. Mol. Cell. Biol. 1994; 14: 8096-8106Crossref PubMed Scopus (633) Google Scholar, 4Rouet P. Smih F. Jasin M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6064-6068Crossref PubMed Scopus (478) Google Scholar, 5Choulika A. Perrin A. Dujon B. Nicolas J.F. Mol. Cell. Biol. 1995; 15: 1968-1973Crossref PubMed Scopus (381) Google Scholar, 6Segal D.J. Carroll D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 806-810Crossref PubMed Scopus (43) Google Scholar, 7Brenneman M. Gimble F.S. Wilson J.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3608-3612Crossref PubMed Scopus (53) Google Scholar), but this approach requires the prior insertion of the recognition sequence into the locus. Because intermolecular triple helices can provoke DNA repair (8Wang G. Seidman M.M. Glazer P.M. Science. 1996; 271: 802-805Crossref PubMed Scopus (299) Google Scholar), oligonucleotide-mediated triple helix formation has been proposed as a potentially more general approach to sensitizing a target site to homologous recombination (9Faruqi A.F. Seidman M.M. Segal D.J. Carroll D. Glazer P.M. Mol. Cell. Biol. 1996; 16: 6820-6828Crossref PubMed Scopus (95) Google Scholar, 10Faruqi A.F. Datta H.J. Carroll D. Seidman M.M. Glazer P.M. Mol. Cell. Biol. 2000; 20: 990-1000Crossref PubMed Scopus (118) Google Scholar, 11Luo Z. Macris M.A. Faruqi A.F. Glazer P.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9003-9008Crossref PubMed Scopus (90) Google Scholar, 12Vasquez K.M. Wilson J.H. Trends. Biochem. Sci. 1998; 23: 4-9Abstract Full Text PDF PubMed Scopus (167) Google Scholar). TFOs1 can bind in the major groove of DNA to polypurine/polypyrimidine sequences, forming specific Hoogsteen or reverse-Hoogsteen hydrogen bonds with the purine strand of the duplex (13Chan P.P. Glazer P.M. J. Mol. Med. 1997; 75: 267-282Crossref PubMed Scopus (216) Google Scholar, 14Praseuth D. Guieysse A.L. Helene C. Biochim. Biophys. Acta. 1999; 1489: 181-206Crossref PubMed Scopus (282) Google Scholar). Triplex formation has been shown to inhibit transcription in mammalian cells (15Faria M. Wood C.D. Perrouault L. Nelson J.S. Winter A. White M.R. Helene C. Giovannangeli C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3862-3867Crossref PubMed Scopus (132) Google Scholar) and can be used to deliver a DNA-reactive conjugate to a specific target site both in complex DNA mixtures in vitro (16Strobel S.A. Doucette-Stamm L.A. Riba L. Housman D.E. Dervan P.B. Science. 1991; 254: 1639-1642Crossref PubMed Scopus (188) Google Scholar, 17Gunther E.J. Havre P.A. Gasparro F.P. Glazer P.M. Photochem. Photobiol. 1996; 63: 207-212Crossref PubMed Scopus (24) Google Scholar) and within mammalian cells in culture (18Giovannangeli C. Diviacco S. Labrousse V. Gryaznov S. Charneau P. Helene C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 79-84Crossref PubMed Scopus (166) Google Scholar, 19Wang G. Levy D.D. Seidman M.M. Glazer P.M. Mol. Cell. Biol. 1995; 15: 1759-1768Crossref PubMed Google Scholar, 20Majumdar A. Khorlin A. Dyatkina N. Lin F.-L.M. Powell J. Liu J. Fei Z. Khripine Y. Watanabe K.A. George J. Glazer P.M. Seidman M.M. Nat. Genet. 1998; 20: 212-214Crossref PubMed Scopus (153) Google Scholar, 21Oh D.H. Hanawalt P.C. Nucleic Acids Res. 1999; 27: 4734-4742Crossref PubMed Scopus (41) Google Scholar, 22Belousov E.S. Afonina I.A. Kutyavin I.V. Gall A.A. Reed M.W. Gamper H.B. Wydro R.M. Meyer R.B. Nucleic Acids Res. 1998; 26: 1324-1328Crossref PubMed Scopus (56) Google Scholar), in some cases leading to site-directed mutations (19Wang G. Levy D.D. Seidman M.M. Glazer P.M. Mol. Cell. Biol. 1995; 15: 1759-1768Crossref PubMed Google Scholar,20Majumdar A. Khorlin A. Dyatkina N. Lin F.-L.M. Powell J. Liu J. Fei Z. Khripine Y. Watanabe K.A. George J. Glazer P.M. Seidman M.M. Nat. Genet. 1998; 20: 212-214Crossref PubMed Scopus (153) Google Scholar). Triplex formation, by itself, can be mutagenic, and evidence suggests that the nucleotide excision repair (NER) and transcription-coupled repair pathways may play a role in the triplex-induced mutagenesis (8Wang G. Seidman M.M. Glazer P.M. Science. 1996; 271: 802-805Crossref PubMed Scopus (299) Google Scholar). In previous work, we found that triple helix-directed psoralen cross-links could stimulate recombination in a plasmid substrate containing two tandem copies of the supFG1 reporter gene (9Faruqi A.F. Seidman M.M. Segal D.J. Carroll D. Glazer P.M. Mol. Cell. Biol. 1996; 16: 6820-6828Crossref PubMed Scopus (95) Google Scholar). Subsequent work established that triplex formation, even in the absence of covalent DNA damage, could stimulate recombination between repeated sequences, an effect that was absent in cells deficient in the NER factor, XPA (10Faruqi A.F. Datta H.J. Carroll D. Seidman M.M. Glazer P.M. Mol. Cell. Biol. 2000; 20: 990-1000Crossref PubMed Scopus (118) Google Scholar). Recent work has extended these findings to the demonstration of TFO-induced recombination at a chromosomal locus containing two tandem copies of the herpes simplex virus thymidine kinase gene, following direct intranuclear microinjection of the oligonucleotides (11Luo Z. Macris M.A. Faruqi A.F. Glazer P.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9003-9008Crossref PubMed Scopus (90) Google Scholar). Based on the ability of TFOs to mediate specific molecular recognition of a DNA target site within a cellular genome and on the observation that triplex formation can stimulate recombination, we also tested a series of bifunctional oligonucleotides consisting of a TFO designed to bind to bp 167–196 of the supFG1 reporter gene coupled to a short (40 nt) segment of DNA homologous to bp 121–160 of the gene. Such a hybrid molecule, designated a tethered donor-TFO (TD-TFO), was found to mediate recombination with the supFG1 gene present in an SV40-based episomal vector in monkey cells at frequencies in the range of 0.1–1% (Ref. 23Chan P.P. Lin M. Faruqi A.F. Powell J. Seidman M.M. Glazer P.M. J. Biol. Chem. 1999; 274: 11541-11548Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar and data not shown), demonstrating that TFOs can promote intermolecular as well asintramolecular recombination in mammalian cells. This result is consistent with studies demonstrating that bifunctional oligonucleotides can mediate both triplex formation and strand invasion on plasmid substrates in vitro (24Gamper H.B. Hou Y.M. Stamm M.R. Podyminogin M.A. Meyer R.B. J. Am. Chem. Soc. 1998; 120: 2182-2183Crossref Scopus (16) Google Scholar, 25Broitman S. Amosova O. Dolinnaya N.G. Fresco J.R. J. Biol. Chem. 1999; 274: 21763-21768Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). In the present study, we have used a plasmid-based assay to investigate triple helix-induced recombination in human cell-free extracts. We find that triple helix formation can stimulate recombination between a plasmid and short homologous fragments in vitro. Stimulation was observed whether or not the donor fragment was directly linked to the TFO. Recombination was reduced in the absence of the TFO as well as when the TFO was substituted with a non-triplex-forming, scrambled sequence oligonucleotide. To probe the mechanism of the induced recombination, the roles of the NER damage recognition factor, XPA (26Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Crossref PubMed Scopus (965) Google Scholar), and the human recombinase, HsRad51 (27Gupta R.C. Bazemore L.R. Golub E.I. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 463-468Crossref PubMed Scopus (239) Google Scholar), were directly tested by experimental manipulation of the respective in the either with specific or supplementation with purified We that both XPA and HsRad51 for triple helix-induced recombination, and that HsRad51 can the of the vector plasmid containing a supFG1 gene with an to point mutation at was P.P. Lin M. Faruqi A.F. Powell J. Seidman M.M. Glazer P.M. J. Biol. Chem. 1999; 274: 11541-11548Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). were by the and purified by either or high pressure liquid chromatography by in oligonucleotides of but were at the to by the of at the In the the segment between the donor fragment and the TFO domain of the sequence in which a specific designated has the sequence and is designed to bind as a strand to bp of the supFG1 gene. donor domain of a DNA fragment homologous to 121–160 of the supFG1 gene scrambled sequence has the base as but at of has been P.M. Mol. Cell. Biol. PubMed Scopus Google Scholar). cells were and by the and were as cell for HsRad51 was purified plasmid of chromatography and have been Golub E.I. G. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar). was by a of following the HsRad51 was into to high specific to the HsRad51 Golub E.I. G. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar). XPA was produced using an vector from containing the human XPA with an in the plasmid was in as by and Wood Wood PubMed Scopus Google Scholar). XPA was purified using chromatography were with a containing and was used to using to the was used to To that the and purified was the and present as in XPA was to both and results a at a molecular of a of the were with the purified XPA to high specific to cell was as P.M. Mol. Cell. Biol. PubMed Scopus Google Scholar). cells were with and in by using a was in cell of and a cell of of was and by by at × for at was in and was in the for was in liquid and at typically of of of pSupFG1/G144C plasmid of oligonucleotides donor or of and of cell-free in a at the were by the of and of at for the plasmid DNA was by and and in of of the was used to by as P.M. Mol. Cell. Biol. PubMed Scopus Google Scholar), by of the cells on indicator for of supFG1 gene as (19Wang G. Levy D.D. Seidman M.M. Glazer P.M. Mol. Cell. Biol. 1995; 15: 1759-1768Crossref PubMed Google Scholar). or were to and with for at were with and with of cell for on with or was by and examined by and used in the in vitro recombination the cell with HsRad51 the were and with of and was and at the was and the was a containing and was directly used to the depleted extracts for the recombination assay substrate for recombination was the plasmid vector containing a of the supFG1 which has an to mutation at bp of this gene can be in indicator an in the gene, and is a reporter of recombination that the gene to the gene also a site at the of the gene to which the TFO can bind to form a triple helix in the In a to promote recombination, we designed a in which the TFO is tethered to a donor DNA fragment homologous to a of the target gene a sequence P.P. Lin M. Faruqi A.F. Powell J. Seidman M.M. Glazer P.M. J. Biol. Chem. 1999; 274: 11541-11548Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). This target site recognition triple helix formation at the the donor fragment for recombination and This also is to the ability of a triple itself, to provoke DNA potentially the of recombination with the homologous donor In the bifunctional molecule, the donor of a of to be homologous to 121–160 of the gene at the sequence that of the gene. In previous work, we the of triplex-induced recombination upon of into monkey cells the pSupFG1/G144C vector as an target P.P. Lin M. Faruqi A.F. Powell J. Seidman M.M. Glazer P.M. J. Biol. Chem. 1999; 274: 11541-11548Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). To investigate the mechanism of triplex-induced recombination, in the present work we have tested the ability of triplex formation to promote recombination within human cell-free extracts. oligonucleotides were with the target pSupFG1/G144C vector in cell extracts with and a the plasmid vector DNA was and used to to for supFG1 gene results that the bifunctional was in the extracts and produced gene reversion at a frequency of × that this effect in the and was not by recombination in the indicator in the were observed upon transformation of the into bacteria. A donor fragment was also as co-mixture of A the pSupFG1/G144C plasmid to a of consistent with the ability of short fragments of DNA to mediate recombination and L. D. Biol. 1989; Google Scholar, Mol. Biol. 2000; Google Scholar, K.M. Mol. Cell. Biol. PubMed Scopus Google Scholar). However, the effect of was 4-fold than that of A demonstrating the of the TFO domain and direct evidence for triplex-induced recombination in vitro. itself, the TFO domain produced reversion over the for the sequence by the A donor the in which and the A donor oligonucleotide were not linked but were as separate with the plasmid substrate also produced an of recombination, at a frequency of × 10−5, as high as that produced by the linked This result evidence that a TFO can stimulate recombination between a donor fragment and a target locus. In the donor fragment in this is separate from the the result a role for the TFO in recombination that is from ability to deliver a tethered donor fragment to the target In the A donor was linked to an oligonucleotide segment designated consisting of the base as but a scrambled sequence not bind to the supFG1 gene and not form a also has to the target gene. of to the donor fragment was found to inhibit recombination to the donor fragment alone. results above establish that triplex-induced recombination can be in cell extracts in vitro. using this in vitro we to the of recombination and repair proteins in the of triplex-induced is a human that in homologous recombination and has been shown to mediate DNA and strand P. Biochem. Sci. 1998; 23: Full Text Full Text PDF PubMed Scopus Google Scholar). To the role of in this homologous gene we used a to HsRad51 from the cell of HsRad51 from the was by depleted was tested for the ability to triplex-induced recombination both in the of the linked bifunctional and in the of the separate A and of HsRad51 was found to the frequency of in both that in the absence of the HsRad51 of HsRad51 on triplex-induced recombination in cell-free results the data from in a new results the data from we tested the to which the triplex-induced recombination in the extracts could be restored by the of HsRad51 of HsRad51 were to the depleted and the recombination assay was the of a of to a of the triplex-induced recombination was We that the of by purified HsRad51 the from the extracts of with HsRad51. To we the extracts with HsRad51 of the to the depleted extracts was found to completely the recombination that HsRad51 more than HsRad51 and that HsRad51 by itself, for the of the This result is not in of evidence that the recombination complex in human cells of and as well as of the and J. Genet. 1999; 15: Full Text Full Text PDF PubMed Scopus Google Scholar, P. K.M. S. Res. 2000; PubMed Scopus Google of HsRad51 supplementation on triplex-induced recombination in cell-free of of of of of HsRad51 of HsRad51 results the data from in a new results the data from of to were to cell and triplex-induced recombination was in the of the linked donor fragment and TFO and the donor TFO HsRad51 was found to the frequency of the triplex-induced In the with of HsRad51 in the was a However, at of of were even by itself, the of the can to cell of HsRad51 supplementation on triplex-induced recombination in cell-free of of of of of of of results the data from in a new results the data from In previous work TFO-induced mutagenesis and recombination within SV40 in human cells, we evidence that the ability of triplex formation to stimulate DNA is dependent on the of the NER (8Wang G. Seidman M.M. Glazer P.M. Science. 1996; 271: 802-805Crossref PubMed Scopus (299) Google Scholar, 10Faruqi A.F. Datta H.J. Carroll D. Seidman M.M. Glazer P.M. Mol. Cell. Biol. 2000; 20: 990-1000Crossref PubMed Scopus (118) Google Scholar). To direct evidence in of this proposed we tested the for the NER damage recognition factor, XPA (26Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Crossref PubMed Scopus (965) Google Scholar), in the triplex-induced recombination in the cell extracts. A was human XPA produced in and was found to a in human cells of the not this XPA was from the extracts by of XPA was by of the of XPA from the extracts was found to the frequency of TFO-induced recombination, whether or not the TFO was linked to the donor fragment both the and the the of XPA reduced the frequency of to that by the donor fragment the ability of a triple helix to stimulate recombination on the XPA This result the that the NER can a triple helix as a DNA that can to recombination or of XPA on triplex-induced recombination in cell-free of of of results the data from in a new results the data from XPA we tested the ability of XPA to the triplex-induced recombination results that of XPA in the depleted extracts. These results establish a direct role for XPA in the ability of a triple helix to stimulate work that triplex-induced recombination can be detected in human cell-free extracts. A TFO that with high to a of the gene within the pSupFG1/G144C vector was found to stimulate recombination between the vector and a donor Recombination was induced both when the donor fragment was linked to the TFO and when was present as a was to in the extracts and not in the indicator were observed the were in the extracts. donor by itself, was to in recombination with the plasmid in the consistent with previous studies that have detected intermolecular recombination in mammalian cell extracts D. A. S. J.M. L. Biol. PubMed Google Scholar). However, the present work that intermolecular recombination can be by strand to of the of TFO-induced recombination of the role of in the of a human homologous to P. Biochem. Sci. 1998; 23: Full Text Full Text PDF PubMed Scopus Google Scholar), from the reduced the of induced but purified HsRad51 not for the When the was and used to the depleted the induced recombination was These results that the in to HsRad51 that for the Such could proteins that proposed to play a role in homologous recombination, as and J. Genet. 1999; 15: Full Text Full Text PDF PubMed Scopus Google Scholar, P. K.M. S. Res. 2000; PubMed Scopus Google Scholar). the the of HsRad51 to the extracts produced an frequency of that itself, a role in the This result is consistent with the observation that HsRad51 can a in the frequency of recombination in reporter gene substrates in mammalian cells 1999; PubMed Scopus Google Scholar). NER damage recognition factor, was also found to play an role in the triplex-induced recombination, as induced were in the extracts XPA of the induced extracts with XPA restored the induced recombination This result not is consistent with previous work that triplex-induced mutagenesis and recombination reduced in human cell deficient in XPA (8Wang G. Seidman M.M. Glazer P.M. Science. 1996; 271: 802-805Crossref PubMed Scopus (299) Google Scholar, 10Faruqi A.F. Datta H.J. Carroll D. Seidman M.M. Glazer P.M. Mol. Cell. Biol. 2000; 20: 990-1000Crossref PubMed Scopus (118) Google Scholar), also directly that XPA is for the the data a in which the oligonucleotide-mediated triple helix is by repair that can Such may either be with of mutation in an homologous DNA is as substrates for repair by a of homologous In the the TFO was found to the plasmid to recombination either with a linked donor fragment or with an at the This ability of the TFO to stimulate recombination between the target site and an fragment is in to a previous TFO-induced recombination in cells, in which the linked was found to be 4-fold more than the of the P.P. Lin M. Faruqi A.F. Powell J. Seidman M.M. Glazer P.M. J. Biol. Chem. 1999; 274: 11541-11548Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In that study, the pSupFG1/G144C vector was into cells, and the cells were the with the the vector DNA was for in indicator bacteria. We this between the previous cell and the present work to in the in vitro the TFO and donor fragment present in whether or not linked in the the ability of the TFO to deliver a linked donor fragment to the target site and in with the of is not as in recombination as is the ability of the triplex to provoke DNA In in the cell both of the TFO to be although to be whether the for linkage of the donor to the TFO in cells can be by the of donor fragment a gene correction in which a TFO could be used in with donor greater than that can be in with the TFO. This be previous studies recombination between episomal or chromosomal targets and in mammalian cells have shown that fragments in the range of bp or of recombination than short fragments in the range used L. D. Biol. 1989; Google Scholar, Mol. Biol. 2000; Google Scholar, K.M. Mol. Cell. Biol. PubMed Scopus Google Scholar, G. Jasin M. P. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar, Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar). the work that triplex-induced recombination can be detected in human cell and into the mechanism by roles for HsRad51 and results a of triplex-induced recombination that on NER and on homologous repair of ability to TFO-induced recombination in vitro as a for of the in which triplex formation can provoke DNA and may in to TFOs to promote in human cells. We Golub and M. Radding for and from the for cells, and for in this We also and S. for

Although many bacterial chromosomes require only one replication initiator protein, e.g., DnaA, most plasmid replicons depend on dual initiators: host-encoded DnaA and plasmid-encoded Rep initiator protein for replication initiation. Using the plasmid pSC101 as a model system, this work investigates the biological rationale for the requirement for dual initiators and shows that the plasmid-encoded RepA specifically interacts with the replicative helicase DnaB. Mutations in DnaB or RepA that disrupt RepA-DnaB interaction cause failure to load DnaB to the plasmid ori in vitro and to replicate the plasmid in vivo. Although, interaction of DnaA with DnaB could not substitute for RepA-DnaB interaction for helicase loading, DnaA along with integration host factor, DnaC, and RepA was essential for helicase loading. Therefore, DnaA is indirectly needed for helicase loading. Instead of a common surface of interaction with initiator proteins, interestingly, DnaB helicase appears to have at least a limited number of nonoverlapping surfaces, each of which interacts specifically with a different initiator protein.

Synthetic triple helix-forming oligodeoxyribonucleotides (TFOs) have been used to alter gene expression and to induce targeted genome modification in cells and animals. However, the efficacy of such oligodeoxyribonucleotides (ODNs) depends on efficient intracellular delivery. A novel vector system was tested for the production of single-stranded DNA (ssDNA) to serve as a TFO in mouse cells. Mouse cells carrying a substrate that can report triplex-stimulated intrachromosomal recombination were transfected with a series of ssDNA vectors, and induced recombination was assayed. Transfection with a vector set designed to generate a 34 nt G-rich ssDNA capable of triplex formation at a 30 bp polypurine target site within the reporter substrate yielded recombinants at a frequency of 196 x 10(-6), versus a background frequency of 45 x 10(-6) in mock transfected cells. No induction was seen when a vector set lacking the TFO sequence insert was tested or when the component vectors were transfected individually. Vectors engineered to express a C-rich 34 nt sequence (not expected to form triplex under physiological conditions) had no effect over background. Primer extension analyses on lysates from transfected cells confirmed the production of the intended ssDNAs. These results suggest that ssDNA molecules of a defined sequence can be generated intracellularly using a novel vector system and that such molecules are active in mediating triplex-dependent chromosomal events. The ability to produce active TFOs within cells may provide a new foundation for triplex-based gene targeting strategies.

Triplex-forming oligonucleotides (TFOs) have the potential to serve as gene therapeutic agents on the basis of their ability to mediate site-specific genome modification via induced recombination. However, high-affinity triplex formation is limited to polypurine/polypyrimidine sites in duplex DNA. Because of this sequence restriction, careful analysis is needed to identify suitable TFO target sites within or near genes of interest. We report here an examination of two key parameters which influence the efficiency of TFO-induced recombination: (1) binding affinity of the TFO for the target site and (2) the distance between the target site and the mutation to be corrected. To test the influence of binding affinity, we compared induced recombination in human cell-free extracts by a series of G-rich oligonucleotides with an identical base composition and an increasing number of mismatches in the third strand binding code. As the number of mismatches increased and, therefore, binding affinity decreased, induced recombination frequency also dropped. There was an apparent threshold at an equilibrium dissociation constant (K(d)) of 1 x 10(-)(7) M. In addition, TFO chemical modification with N,N-diethylethylenediamine (DEED) internucleoside linkages to confer improved binding was found to yield increased levels of induced recombination. To test the ability of triplex formation to induce recombination at a distance, episomal targets with informative reporter genes were constructed to contain polypurine TFO target sites at varying distances from the mutations to be corrected. TFO-induced recombination in mammalian cells between a plasmid vector and a donor oligonucleotide was detected at distances ranging from 24 to 750 bp. Together, these results indicate that TFO-induced recombination requires high-affinity binding but can affect sites hundreds of base pairs away from the position of triplex formation.