Distance and Affinity Dependence of Triplex-Induced Recombination

Knauert, Melissa P.; Lloyd, Janice A.; Rogers, Faye A.; Datta, Hirock J.; Bennett, Michael L.; Weeks, Daniel L.; Glazer, Peter M.

doi:10.1021/bi0481040

Cited by 31 publications

(30 citation statements)

References 35 publications

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“…From the data presented in Figure 9 it is seen that target binding of PNA3051 did not using the oligonucleotide donor. These results parallel previous findings 28,29 showing that unconjugated (deoxyribosephophate) TFOs can stimulate the correction of nearby mutations using single-stranded oligonucleotide donors.…”

Section: Effects Of Triplex Pnas In An Ex Vivo Cellular Assaysupporting

confidence: 91%

Targeted gene correction using psoralen, chlorambucil and camptothecin conjugates of triplex forming peptide nucleic acid (PNA)

Birkedal¹,

Nielsen²

2011

Artificial DNA: PNA & XNA

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Section: Effects Of Triplex Pnas In An Ex Vivo Cellular Assaysupporting

confidence: 91%

Targeted gene correction using psoralen, chlorambucil and camptothecin conjugates of triplex forming peptide nucleic acid (PNA)

Birkedal¹,

Nielsen²

2011

Artificial DNA: PNA & XNA

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“…Due to the small number of sequences analyzed in each of these studies, the pattern was not detected. Polypurine (pPu) tracts and their complementary pPy tracts are believed to stimulate recombination (4,(21)(22)(23). They can form triplex DNA, which can stabilize strand invasion events (21)(22)(23).…”

Section: Discussionmentioning

confidence: 99%

“…Polypurine (pPu) tracts and their complementary pPy tracts are believed to stimulate recombination (4,(21)(22)(23). They can form triplex DNA, which can stabilize strand invasion events (21)(22)(23). In addition, pPy tracts as short as 18 bp have been demonstrated to promote oligonucleotide-directed gene correction at least 23 bp away in vivo and strand invasion (D-loop formation) at sites up to 4 kb away in vitro (4).…”

Section: Discussionmentioning

confidence: 99%

Preferential Integration of Adeno-Associated Virus Type 2 into a Polypyrimidine/Polypurine-Rich Region within AAVS1

McAlister

Owens

2007

J Virol

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Adeno-associated virus type 2 (AAV2) preferentially integrates its genome into the AAVS1 locus on human chromosome 19. Preferential integration requires the AAV2 Rep68 or Rep78 protein (Rep68/78), a Rep68/78 binding site (RBS), and a nicking site within AAVS1 and may also require an RBS within the virus genome. To obtain further information that might help to elucidate the mechanism and preferred substrate configurations of preferential integration, we amplified junctions between AAV2 DNA and AAVS1 from AAV2-infected HeLaJW cells and cells with defective Artemis or xeroderma pigmentosum group A genes. We sequenced 61 distinct junctions. The integration junction sequences show the three classical types of nonhomologous-endjoining joints: microhomology at junctions (57%), insertion of sequences that are not normally contiguous with either the AAV2 or the AAVS1 sequences at the junction (31%), and direct joining (11%). These junctions were spread over 750 bases and were all downstream of the Rep68/78 nicking site within AAVS1. Two-thirds of the junctions map to 350 bases of AAVS1 that are rich in polypyrimidine tracts on the nicked strand. The majority of AAV2 breakpoints were within the inverted terminal repeat (ITR) sequences, which contain RBSs. We never detected a complete ITR at a junction. Residual ITRs at junctions never contained more than one RBS, suggesting that the hairpin form, rather than the linear ITR, is the more frequent integration substrate. Our data are consistent with a model in which a cellular protein other than Artemis cleaves AAV2 hairpins to produce free ends for integration.Adeno-associated virus type 2 (AAV2) is a naturally defective human parvovirus that usually requires a helper virus, such as an adenovirus or a herpesvirus, for productive infection (41). In the absence of helper virus, AAV2 can establish a latent infection by episomal persistence (54) or by integrating its DNA into the host chromosomes, preferentially within a 4-kb region of human chromosome 19 designated AAVS1 (11). This is not site-specific integration in the classic sense, but approximately 70% of integration events occur somewhere within this locus (Fig. 1A) (20,26,51). The mechanism is unknown, but it does require the Rep68 or Rep78 (Rep68/78) protein encoded by AAV2 and a 33-bp region of AAVS1 (Fig. 1A) that includes a Rep68/78 binding site (RBS) and a nicking site for Rep68/78 that resembles the terminal resolution site (trs) within the 145-base inverted terminal repeats (ITRs) of the AAV2 genome (Fig. 2) (29,57,64,67). The trs got its name because of its role in AAV2 replication. AAV2 has a linear, single-stranded DNA genome (4,679 bases; GenBank accession no. AF043303), and the ITRs are essential for replication. The ITRs are palindromic and fold into T-shaped hairpin structures, one of which provides a 3Ј end that primes secondstrand synthesis by a cellular DNA polymerase (Fig. 2) (10, 65). The hairpin is then nicked by Rep68/78 at trs (Fig. 2) and unwound, and the end is replicated. As a result of this mo...

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“…The plucTC18 vector was derived from a plasmid constructed by subcloning the firefly luciferase gene Fluc+ (pGL3-Basic Vector; Promega) into pcDNA5/FRT (Invitrogen) (25). The TC18 target site was inserted 40 bp upstream of the Fluc+ start site.…”

Section: Methodsmentioning

confidence: 99%

Triplex-induced recombination and repair in the pyrimidine motif

Kalish

Seidman²,

Weeks³

et al. 2005

Nucleic Acids Research

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Triplex-forming oligonucleotides (TFOs) bind DNA in a sequence-specific manner at polypurine/polypyrimidine sites and mediate targeted genome modification. Triplexes are formed by either pyrimidine TFOs, which bind parallel to the purine strand of the duplex (pyrimidine, parallel motif), or purine TFOs, which bind in an anti-parallel orientation (purine, anti-parallel motif). Both purine and pyrimidine TFOs, when linked to psoralen, have been shown to direct psoralen adduct formation in cells, leading to mutagenesis or recombination. However, only purine TFOs have been shown to mediate genome modification without the need for a targeted DNA-adduct. In this work, we report the ability of a series of pyrimidine TFOs, with selected chemical modifications, to induce repair and recombination in two distinct episomal targets in mammalian cells in the absence of any DNA-reactive conjugate. We find that TFOs containing N3′→P5′ phosphoramidate (amidate), 5-(1-propynyl)-2′-deoxyuridine (pdU), 2′-O-methyl-ribose (2′-O-Me), 2′-O-(2-aminoethyl)-ribose, or 2′-O, 4′-C-methylene bridged or locked nucleic acid (LNA)-modified nucleotides show substantially increased formation of non-covalent triplexes under physiological conditions compared with unmodified DNA TFOs. However, of these modified TFOs, only the amidate and pdU-modified TFOs mediate induced recombination in cells and stimulate repair in cell extracts, at levels comparable to those seen with purine TFOs in similar assays. These results show that amidate and pdU-modified TFOs can be used as reagents to stimulate site-specific gene targeting without the need for conjugation to DNA-reactive molecules. By demonstrating the potential for induced repair and recombination with appropriately modified pyrimidine TFOs, this work expands the options available for triplex-mediated gene targeting.

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Distance and Affinity Dependence of Triplex-Induced Recombination

Cited by 31 publications

References 35 publications

Targeted gene correction using psoralen, chlorambucil and camptothecin conjugates of triplex forming peptide nucleic acid (PNA)

Targeted gene correction using psoralen, chlorambucil and camptothecin conjugates of triplex forming peptide nucleic acid (PNA)

Preferential Integration of Adeno-Associated Virus Type 2 into a Polypyrimidine/Polypurine-Rich Region within AAVS1

Triplex-induced recombination and repair in the pyrimidine motif

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