DNA mismatch repair detected in human cell extracts.

Glazer, Peter M.; Sarkar, Saumyen N.; Chisholm, G E; Summers, William C.

doi:10.1128/mcb.7.1.218

Cited by 40 publications

(25 citation statements)

References 24 publications

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“…The shuttle vector plasmid pSupFG1͞ G144C, containing a supFG1 gene with an inactivating G:C to C:G point mutation at position 144, has been described (11). Construction of Escherichia coli SY302 lacZ125 (Am) recA56 hsdR2::Tn10 trp-49 has been described (26). HeLa cells were maintained and grown by the National Cell Culture Center (Minneapolis) and were obtained as frozen cell pellets for extract preparation.…”

Section: Methodsmentioning

confidence: 99%

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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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Section: Methodsmentioning

confidence: 99%

“…Cell-free extracts were prepared as described (25,26). Recombination reactions consisting of 3 g of pSupFG1͞G144C plasmid DNA and 3 g each of selected oligonucleotide or PNA were carried out as described (25).…”

Section: Methodsmentioning

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

View full text Add to dashboard Cite

show abstract

“…Cells-Construction of E. coli SY302 lacZ125(Am) recA56 hsdR2::Tn10 trp-49 has been described previously (28). HeLa cells were maintained and grown by the National Cell Culture Center (Minneapolis, MN) and were obtained as cell pellets for extract preparation.…”

Section: Methodsmentioning

confidence: 99%

“…Preparation of Cell-free Extract-HeLa whole cell extract was prepared as described previously (28). Briefly, HeLa cells were washed with phosphate-buffered saline and resuspended in 0.01 M Tris-HCl, pH 7.9, 1 mM EDTA, 5 mM DTT, followed by lysis using a Dounce homogenizer.…”

Section: Methodsmentioning

confidence: 99%

Triplex-induced Recombination in Human Cell-free Extracts

Datta

Chan

Vásquez

et al. 2001

Journal of Biological Chemistry

100

102

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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.

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“…In Vitro Repair Assays-HeLa cell-free extracts were prepared as described previously (24). Oligonucleotides pso-AG10 and pso-AG30 were 3Ј end-labeled using terminal transferase and [␣- 32 P]dideoxy-ATP (Amersham).…”

Section: Methodsmentioning

confidence: 99%

Altered Repair of Targeted Psoralen Photoadducts in the Context of an Oligonucleotide-mediated Triple Helix

Wang

Glazer

1995

Journal of Biological Chemistry

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Oligonucleotides can bind as third strands of DNA in a sequence-specific manner to form triple helices. Psoralen-conjugated, triplex-forming oligonucleotides (TFOs) have been used for the site-specific modification of DNA to inhibit transcription and to target mutations to selected genes. Such strategies, however, must take into account the ability of the cell to repair the triplexdirected lesion. We report experiments showing that the pattern of mutations produced by triplex-targeted psoralen adducts in an SV40 shuttle vector in monkey COS cells can be influenced by the associated third strand. Mutations induced by psoralen adducts in the context of a TFO of length 10 were the same as those generated by isolated adducts but were found to be different from those generated in the presence of a TFO of length 30 at the same target site. In complementary experiments, HeLa whole cell extracts were used to directly assess repair of the TFO-directed psoralen adducts in vitro. Excision of the damaged DNA was inhibited in the context of the 30-mer TFO, but not the 10-mer. These results suggest that an extended triple helix of length 30, which exceeds the typical size of the nucleotide excision repair patch in mammalian cells, can alter repair of an associated psoralen adduct. We present a model correlating these results and proposing that the incision steps in nucleotide excision repair in mammalian cells can be blocked by the presence of a third strand of sufficient length and binding affinity, thereby changing the pattern of mutations. These results may have implications for the use of triplex-forming oligonucleotides for genetic manipulation, and they may lead to the use of such oligonucleotides as tools to probe DNA repair pathways.Oligonucleotides can bind in the major groove of duplex DNA to form triple helices in a sequence-specific manner (1-4). Progress in elucidating the third strand binding code has raised the possibility of developing nucleic acids as sequencespecific reagents for research and possibly therapeutic applications. Oligonucleotide-mediated triplex formation has been shown to inhibit transcription factor binding to promoter sites and to block transcription in vitro and in vivo (5). Strategies to enhance transcription inhibition using oligonucleotide-intercalator conjugates have also been employed (6, 7). Triplex formation has further been used as a tool to generate unique cleavage sites in DNA in vitro (8). We and others have explored the use of triplex-forming oligonucleotides (TFOs) 1 as a method to deliver a tethered mutagen to a selected gene for the site-specific introduction of DNA damage (9 -12). Psoralen-conjugated oligonucleotides directed to a site in a mutation reporter gene were found to induce targeted mutations in (10) and SV40 (9) DNA in experiments in which the triplex formation was carried out in vitro. In recent work, conditions were determined under which targeted mutagenesis of an SV40 vector could be achieved in vivo via intracellular triplex formation using psoralen-linked TF...

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DNA mismatch repair detected in human cell extracts.

Cited by 40 publications

References 24 publications

Site-directed recombination via bifunctional PNA–DNA conjugates

Site-directed recombination via bifunctional PNA–DNA conjugates

Triplex-induced Recombination in Human Cell-free Extracts

Altered Repair of Targeted Psoralen Photoadducts in the Context of an Oligonucleotide-mediated Triple Helix

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