Preparation and Properties of Oligodeoxynucleotides Containing 5-Iodouracil and 5-Bromo- and 5-Iodocytosine

Ferrer, Elisenda; Wiersma, Marten; Kazimierczak, Bernard; Müller, Christoph W.; Eritja, Ramón

doi:10.1021/bc970042l

Cited by 23 publications

(26 citation statements)

References 25 publications

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“…The new derivative is easily prepared and can be removed in mild conditions. Contrary to other brominated nucleosides 21,22 , 8-bromo-dG was found to be very stable to ammonia deprotection. Therefore, the use of more labile groups for the natural bases is not required.…”

Section: Resultsmentioning

confidence: 60%

“…Sequences carrying 5-bromocytosine (E and F) were prepared using tbutylphenoxyacetyl (TBPA)-protected phosphoramidites and deprotected with concentrated ammonia at room temperature to avoid the formation of 5-aminocytosine. 21 Sequences B, C and D prepared with standard phosphoramidites were treated with concentrated ammonia overnight at 50ºC. Special attention was paid to detect the decomposition of 8-bromoguanine to 8-aminoguanine or 8-oxoguanine as it has been described for 5-bromo-pyrimidines 21 and 8-bromo-dA 22 when using hot ammonia solutions for the removal of protecting groups.…”

Section: Preparation Of Oligonucleotides Oligonucleotidesmentioning

confidence: 99%

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Synthesis and Properties of Oligonucleotides Containing 8-Bromo-2′-Deoxyguanosine

Fàbrega

Macías

Eritja

2001

Nucleosides, Nucleotides and Nucleic Acids

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Section: Resultsmentioning

confidence: 60%

Section: Preparation Of Oligonucleotides Oligonucleotidesmentioning

confidence: 99%

Synthesis and Properties of Oligonucleotides Containing 8-Bromo-2′-Deoxyguanosine

Fàbrega

Macías

Eritja

2001

Nucleosides, Nucleotides and Nucleic Acids

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“…The phosphoramidite for 5-iodo-2Ј-deoxyuridine was purchased from Glen Research. Due to the base lability of 5-iodo-2Ј-deoxyuridine (26), the syntheses were performed with Expedite phosphoramidites (Perseptive Biosystems) containing the more labile tert-butylphenoxyacetyl protection group and allowing deprotection with concentrated ammonia at room temperature for 2 h. Purification, evaluation, and hybridization of ODNs were performed as described before (12). Sequences of synthesized ODNs are listed in Table I.…”

Section: Methodsmentioning

confidence: 99%

Identification of the Binding Site for the Extrahelical Target Base in N 6-Adenine DNA Methyltransferases by Photo-cross-linking with Duplex Oligodeoxyribonucleotides Containing 5-Iodouracil at the Target Position

Holz

Dank

Eickhoff

et al. 1999

Journal of Biological Chemistry

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DNA methyltransferases (Mtases) 1 are a biologically important class of enzymes that catalyze the transfer of the activated methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to the N-6 nitrogen of adenine and the N-4 nitrogen of cytosine or the C-5 carbon of cytosine within their DNA recognition sequences (1). As DNA methylation is a postreplicative process that depends on the presence or regulation of DNA Mtases, a particular DNA sequence may exist in its fully methylated, its unmethylated, or transiently in its hemimethylated form. Thus, DNA methylation can be regarded as an increase of the information content of DNA (2), which serves a wide variety of biological functions, including protection of the host genome from endogenous restriction endonucleases, DNA mismatch repair after replication, regulation of gene expression and DNA replication, embryonic development, genomic imprinting, and X-chromosome inactivation (3-5). Furthermore, it plays a role in carcinogenesis (6). Three-dimensional structures are available for the C 5 -cytosine DNA Mtases M.HhaI (7) and M.HaeIII (8) in complex with DNA. Both enzymes consist of two domains forming a positively charged cleft, which accommodates the DNA. However, the most striking feature of these protein-DNA complexes is that the target cytosines are completely rotated out of the DNA double helix and placed in a pocket within the cofactor binding domains, where catalysis takes place. In addition, the structures of the N 6 -adenine DNA Mtases M.TaqI (9) and the N 4 -cytosine DNA Mtase M.PvuII (10) in the absence of DNA were reported. These N 6 -adenine and N 4 -cytosine DNA Mtases (N-DNA Mtases) also have a bilobal structure forming a positively charged cleft. Modeling B-DNA in this cleft showed that the distance between the target base and the bound AdoMet is too large for a direct methyl group transfer, and a base flipping mechanism as observed for the C 5 -cytosine DNA Mtases was postulated. Biochemical evidence for a base flipping mechanism of the N 6 -adenine DNA Mtases M.EcoRI (11) and M.TaqI (12) was obtained using duplex oligodeoxyribonucleotides (ODNs) containing the fluorescent base analogue 2-aminopurine at the target positions. In addition, tighter binding of the * This work was supported by a grant from the Deutsche Forschungsgemeinschaft (We1453/3-2). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.

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“…3). Second, substitution of iodo-UMP at three positions within the 3Ј-proximal region, which is predicted to increase the stability of RNA-DNA hybrid (Ferrer et al 1997), does not reduce the efficiency of −7 oligonucleotide-stimulated transcript release (I. Artsimovitch and R. Landick, unpubl.). However, because transcript release at the pause requires high salt, even with the −7 antisense oligonucleotide, there must be an additional destabilizing force at a -independent terminator.…”

Section: Role Of Rna Hairpin In Transcription Terminationmentioning

confidence: 99%

Interaction of a nascent RNA structure with RNA polymerase is required for hairpin-dependent transcriptional pausing but not for transcript release

Artsimovitch¹,

Landick²

1998

Genes Dev.

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Nascent RNA structures may regulate RNA chain elongation either directly through interaction with RNA polymerase or indirectly by disrupting nascent RNA contacts with polymerase or DNA. To distinguish these mechanisms we tested whether the effects of the his leader pause RNA hairpin could be mimicked by pairing of antisense DNA or RNA oligonucleotides to the nascent transcript. The his pause hairpin inhibits nucleotide addition when it forms 11 nucleotides from the transcript 3 end. It also can terminate transcription when base changes extend its stem to р8 nucleotides from the 3 end. All oligonucleotides that disrupted the pause hairpin reduced the dwell time of RNA polymerase at the pause site dramatically, even when they mimicked the 11-nucleotide 3-proximal RNA spacing or created a suitably positioned RNA loop. Oligonucleotides that paired р8 nucleotides from the pause RNA 3 end could trigger transcript release, but only when added to an already paused complex. These results argue that direct interaction of a nascent RNA hairpin with RNA polymerase delays escape from a pause, but that indirect effects of a hairpin may trigger transcript release from a paused complex. Resistance of the paused complex to pyrophosphorolysis and its reversal by antisense oligonucleotides further suggest that interaction of the pause hairpin with RNA polymerase disengages the RNA 3 end from the active site.[Key Words: RNA polymerase; RNA hairpins; pausing; termination] Received June 2, 1998; revised version accepted August 7, 1998. Nascent RNA hairpins are components of certain classes of intrinsic pause and termination signals that regulate RNA chain elongation (traditionally called -independent terminators and hairpin-dependent pause sites; for review, see Richardson and Greenblatt 1996;. Either pausing or termination can be stimulated by these RNA structures, depending on their distance from the pause or terminated RNA 3Ј end and the surrounding RNA and DNA sequences . A central question about the effects of nascent RNA hairpins is whether they influence transcription through direct interactions with RNA polymerase (RNAP) or if their principal effects on the transcription complex (TC) are indirect and reflect removal of the nascent RNA chain from other interactions.At the well-characterized his leader pause site, a nascent RNA hairpin is one of four components of a multipartite pause signal that directs pausing in a two-step mechanism (Fig. 1). The pause signal consists of the 5-bp stem, 8-nucleotide loop pause hairpin, the 11-nucleotide 3Ј-proximal region between the pause hairpin and the pause RNA 3Ј end, the two bases in the active site, and the DNA duplex downstream from the active site (Chan and Landick 1993). In the first step of the mechanism, isomerization to a paused conformation competes with bypass of the pause site. In the second step, the paused RNAP escapes slowly back to a rapidly elongating conformation by nucleotide addition. The isomerization versus bypass competition is controlled principally by the 3Ј-proxi...

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Preparation and Properties of Oligodeoxynucleotides Containing 5-Iodouracil and 5-Bromo- and 5-Iodocytosine

Cited by 23 publications

References 25 publications

Synthesis and Properties of Oligonucleotides Containing 8-Bromo-2′-Deoxyguanosine

Synthesis and Properties of Oligonucleotides Containing 8-Bromo-2′-Deoxyguanosine

Identification of the Binding Site for the Extrahelical Target Base in N 6-Adenine DNA Methyltransferases by Photo-cross-linking with Duplex Oligodeoxyribonucleotides Containing 5-Iodouracil at the Target Position

Interaction of a nascent RNA structure with RNA polymerase is required for hairpin-dependent transcriptional pausing but not for transcript release

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