Recombination Induced by Triple-Helix-Targeted DNA Damage in Mammalian Cells

Faruqi, A. Fawad; Seidman, Michael M.; Segal, David J.; Carroll, Dana; Glazer, Peter M.

doi:10.1128/mcb.16.12.6820

Cited by 105 publications

(100 citation statements)

References 52 publications

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“…Psoralen cross-links also stimulate recombination (27)(28)(29). Consequently, we were interested in examining the entry of the targeted cross-link site into HDR pathways.…”

mentioning

confidence: 99%

Targeted Gene Knock In and Sequence Modulation Mediated by a Psoralen-linked Triplex-forming Oligonucleotide*

Majumdar

Muniandy

Liu

et al. 2008

Journal of Biological Chemistry

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Information from exogenous donor DNA can be introduced into the genome via homology-directed repair (HDR) pathways. These pathways are stimulated by double strand breaks and by DNA damage such as interstrand cross-links. We have employed triple helix-forming oligonucleotides linked to psoralen (pso-TFO) to introduce a DNA interstrand cross-link at a specific site in the genome of living mammalian cells. Co-introduction of duplex DNA with target region homology resulted in precise knock in of the donor at frequencies 2-3 orders of magnitude greater than with donor alone. Knock-in was eliminated in cells deficient in ERCC1-XPF, which is involved in recombinational pathways as well as cross-link repair. Separately, single strand oligonucleotide donors (SSO) were co-introduced with the pso-TFO. These were 10-fold more active than the duplex knock-in donor. SSO efficacy was further elevated in cells deficient in ERCC1-XPF, in contrast to the duplex donor. Resected single strand ends have been implicated as critical intermediates in sequence modulation by SSO, as well as duplex donor knock in. We asked whether there would be a competition between the donor species for these ends if both were present with the pso-TFO. The frequency of duplex donor knock in was unaffected by a 100-fold molar excess of the SSO. The same result was obtained when the homing endonuclease I-SceI was used to initiate HDR at the target site. We conclude that the entry of double strand breaks into distinct HDR pathways is controlled by factors other than the nucleic acid partners in those pathways. Double strand breaks (DSBs)2 are among the most dangerous forms of DNA damage and may result in deletion, rearrangement of chromosomal sequences, or, if unrepaired, chromosome loss and possibly cell death (1). There are multiple pathways for DSB repair that are distinguished by the identity of the proteins and enzymes involved and their potential for mutagenesis of the break site (2-8). Non-homologous end joining, the major pathway in mammalian cells, is homology-independent and often results in small deletions at the site of the break. There are two homology-directed repair (HDR) pathways in which resected single-stranded ends interact with homologous sequences. In single strand annealing (SSA), the single strand end anneals with a complementary single strand. SSA between two direct repeated sequences results in the retention of one copy of the repeat and deletion of the other copy and the intervening sequence. Homologous recombination repair involves invasion of a DNA duplex by a single strand end and can be error-free or mutagenic depending on the sequence of the invaded duplex. Because the sequences with which they interact need not be absolutely identical to the single strand end, the homology-directed pathways provide an opportunity to manipulate the sequence of the genome.Gene conversion and recombination with exogenous double and single strand donor DNAs occur at impractically low frequencies in mammalian cells (9). However, DSBs in the genom...

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“…Psoralen cross-links also stimulate recombination (27)(28)(29). Consequently, we were interested in examining the entry of the targeted cross-link site into HDR pathways.…”

mentioning

confidence: 99%

Targeted Gene Knock In and Sequence Modulation Mediated by a Psoralen-linked Triplex-forming Oligonucleotide*

Majumdar

Muniandy

Liu

et al. 2008

Journal of Biological Chemistry

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“…Various strategies have been developed to achieve therapeutic targeted gene repair, including small fragment homologous sequence 1,2 ssODN, [3][4][5][6] triple helixforming oligonucleotides containing a reactive group, [7][8][9] bifunctional oligonucleotides based on a triple helixforming DNA recognition domain 10,11 and branched oligonucleotides. 12 Although a general proof of principle for oligonucleotide-based sequence alteration appears to be established, the paradigm for oligonucleotide-based genome alteration remain largely unknown.…”

mentioning

confidence: 99%

Implications of cell cycle progression on functional sequence correction by short single-stranded DNA oligonucleotides

Olsen¹,

Randøl²,

Krauß³

2005

Gene Ther

106

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Oligonucleotide-based sequence alteration in living cells is a substantial methodological challenge in gene therapy. Here, we demonstrate that using corrective single-stranded oligonucleotides (ssODN), high and reproducible sequence correction rates can be obtained. CHO cell lines with chromosomally integrated multiple copy EGFP reporter genes routinely show rates of 4.5% targeted sequence correction after transfection with ssODN. We demonstrate that the cell cycle influences the rates of targeted sequence correction in vivo, with a peak in the early S phase during ssODN exposure. After cell division, the altered genomic sequence is predominantly passed to one daughter cell, indicating that targeted sequence alteration occurs after the replication fork has passed over the targeted site. Although high initial correction rates can be obtained by this method, we show that a majority of the corrected cells arrest in the G2/M cell cycle phase, although 1-2% of the corrected cells form viable colonies. The G2/M arrest observed after targeted sequence correction can be partially released by caffeine, pentoxifylline or Gö 6976 exposure. Despite substantial remaining challenges, targeted sequence alteration based on ssODN increasingly promises to become a powerful tool for functional gene alterations. Gene Therapy (2005) Various strategies have been developed to achieve therapeutic targeted gene repair, including small fragment homologous sequence 1,2 ssODN, 3-6 triple helixforming oligonucleotides containing a reactive group, 7-9 bifunctional oligonucleotides based on a triple helixforming DNA recognition domain 10,11 and branched oligonucleotides. 12 Although a general proof of principle for oligonucleotide-based sequence alteration appears to be established, the paradigm for oligonucleotide-based genome alteration remain largely unknown. 6 The central problem in studying this phenomenon are low rates of in vivo sequence correction rates in cell culture. Generally, observed sequence alteration rates on in vivo chromosomal templates in selected cell lines are in the range of 0.1%, 6 while correction rates in untransformed primary cells are substantially lower. [13][14][15] In the absence of a suitable selection system, such low rates allow only limited functional analysis, although Yoon and co-workers have recently suggested a selection system based on simultaneous targeting of two loci. 16 A further problem is the variety of model systems and assays that were used to measure targeted sequence correction which lead to confusing and often conflicting results. 6 Despite these obstacles, several consistent observations have been made: targeted sequence correction depends centrally on transcription, and ssODN that are complementary to the nontranscribed strand show in most cases substantially higher correction rates than ssODN complementary to the transcribed strand in both episomal and chromosomal assays. 17,18 The involvement of transcription-coupled repair has therefore been suggested. Furthermore, the absence of MSH2, ...

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“…TFOs have been used successfully to inhibit transcription (4), damage DNA through the delivery of a mutagen (5)(6)(7)(8), and provoke mutagenesis through high-affinity binding (9,10). Triple helix formation has also been shown to stimulate recombination in mammalian cells in both episomal and chromosomal targets (11)(12)(13)(14).…”

mentioning

confidence: 99%

Site-directed recombination via bifunctional PNA–DNA conjugates

Rogers

Vásquez

Egholm

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

111

123

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Site-specific DNA binding molecules offer the potential for genetic manipulation of mammalian cells. Peptide nucleic acids (PNAs) are a DNA mimic in which the purine and pyrimidine bases are attached to a polyamide backbone. PNAs bind with high affinity to singlestranded DNA via Watson-Crick base pairing and can form triple helices via Hoogsteen binding to DNA͞PNA duplexes. Dimeric bisPNAs capable of both strand invasion and triplex formation can form clamp structures on target DNAs. As a strategy to promote sitedirected recombination, a bis-PNA was coupled to a 40-nt donor DNA fragment homologous to an adjacent region in the target gene. The PNA-DNA conjugate was found to mediate site-directed recombination with a plasmid substrate in human cell-free extracts, resulting in correction of a mutation in a reporter gene at a frequency at least 60-fold above background. Induced site-specific recombination was also seen when the bis-PNA and the donor DNA were co-mixed without covalent linkage. In addition, the bis-PNA and the bis-PNA-DNA conjugate were found to induce DNA repair specifically in the target plasmid. Both the PNA-induced recombination and the PNAinduced repair were found to be dependent on the nucleotide excision repair factor, XPA (xeroderma pigmentosum complementation group A protein). These results suggest that the formation of a PNA clamp on duplex DNA creates a helical distortion that strongly provokes DNA repair and thereby sensitizes the target site to recombination. The ability to promote recombination in a site-directed manner using PNA-DNA conjugates may provide a useful strategy to achieve targeted correction of defective genes.triple helix ͉ DNA repair

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Recombination Induced by Triple-Helix-Targeted DNA Damage in Mammalian Cells

Cited by 105 publications

References 52 publications

Targeted Gene Knock In and Sequence Modulation Mediated by a Psoralen-linked Triplex-forming Oligonucleotide*

Targeted Gene Knock In and Sequence Modulation Mediated by a Psoralen-linked Triplex-forming Oligonucleotide*

Implications of cell cycle progression on functional sequence correction by short single-stranded DNA oligonucleotides

Site-directed recombination via bifunctional PNA–DNA conjugates

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