Nuclease resistance of an extraordinarily thermostable mini-hairpin DNA fragment, d(GCGAAGC) and its application to<i>in vitro</i>protein synthesis

Yoshizawa, Satoko; Ueda, Takuya; Ishido, Yoshiharu; Miura, Koryo; Watanabe, Kimitsuna; Hirao, Ichiro

doi:10.1093/nar/22.12.2217

Cited by 57 publications

(44 citation statements)

References 24 publications

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“…This conformation explains the unusual stability and the lack of reactivity of template-strand hairpin loops to single-stranded DNA-specific reagents (this paper and ref. 35).…”

Section: Resultsmentioning

confidence: 99%

Supercoil-induced extrusion of a regulatory DNA hairpin

Dai

Greizerstein

Nadas-Chinni

et al. 1997

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

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Bacteriophage N4 virion RNA polymerase (N4 vRNAP) promoters contain inverted repeats, which form a 5-to 7-base-pair stem, 3-base loop hairpin that is required for vRNAP recognition. We show that, contrary to certain theoretical predictions, hairpin extrusion can occur at physiological superhelical densities in a Mg 2؉ -dependent manner. Specific sequences on the template strand are required for hairpin extrusion. These sequences define stable DNA hairpins that are relatively unreactive to single strand-specific probes. In addition, a specific stable hairpin-inducing sequence can regulate transcription in vivo. Thus, a DNA structure, in its natural environment, is involved in transcriptional regulation.DNA supercoiling affects transcription by facilitating or inhibiting RNA polymerase (RNAP) binding and͞or formation of the open complex (1-3). In addition, by stabilizing DNA bending and͞or looping (4-7), supercoiling enhances or inhibits the interaction of activators or repressors with the transcriptional machinery. Supercoiling also promotes the formation of noncanonical DNA structures such as cruciforms, Z-DNA, or triple helices (8) which can affect transcription in vitro (9-11). In no instance, however, is there convincing evidence that such structures play a role in vivo in regulating transcription (reviewed in ref. 12).The three bacteriophage N4 early promoters utilized by the virion (v)RNAP share sequence from Ϫ18 to ϩ1 containing small inverted repeats centered at Ϫ12 (ref. 13). On singlestranded DNA templates, these promoters are utilized efficiently by N4 vRNAP (13,14). Mutational analyses of the promoter template strands indicated that specific sequences and a 5-to 7-base-pair stem, 3-base loop hairpin are important for transcriptional activity (14). Transcription on doublestranded DNA requires supercoiling and Escherichia coli single-stranded DNA-binding protein (EcoSSB) (15). On the basis of these results, we proposed a model in which negative supercoiling extrudes a hairpin to yield an active promoter conformation that is recognized by N4 vRNAP (14). On the contrary, the current theory of the energetics of hairpin extrusion predicts that such small hairpins will extrude only at high, unphysiological superhelical densities (16,17). Here, we show that extrusion of N4 early promoter hairpins occurs at physiological superhelical densities, that this event requires Mg 2ϩ and specific sequence, and that extrusion occurs in vivo. MATERIALS AND METHODS Preparation of Circles Containing N4 Early Promoters.Cloning of the 2.2-kb SpeI-PstI fragment, containing the N4 early promoters P1 and P2 followed by their respective terminators t1 and t2, into pKB652 to yield pXD102, was as described (18). To generate mutant P1 promoters, pXD102 was digested with SmaI and partially digested with NsiI; the 4844-bp fragment lacking the P1 promoter was ligated to the M13 mp11 SmaI-PstI fragment carrying the wild-type or mutant P1 promoters (14). In vivo generation of circles, isolation, preparation of topoisomers, and d...

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“…This conformation explains the unusual stability and the lack of reactivity of template-strand hairpin loops to single-stranded DNA-specific reagents (this paper and ref. 35).…”

Section: Resultsmentioning

confidence: 99%

Supercoil-induced extrusion of a regulatory DNA hairpin

Dai

Greizerstein

Nadas-Chinni

et al. 1997

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

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“…Sequence motifs such as d(GCGN 1-2 AGC) (4) and palindromic d(CGC-GATATCGCG) (5) sequences are well known to form stable hairpin structures, thereby leading to abnormal mobility during gel electrophoresis. These hairpin structures have unique structural properties (5) and have been proposed to play a biological role in the regulation of various cellular events, such as replication, transcription, or RNA processing (6), and have also been reported to be more tolerant to nuclease and heat exposure (7). Different approaches have been described to determine the sequence of strong secondary structures (8 -18), although no general solution has been presented.…”

mentioning

confidence: 99%

Analyses of Secondary Structures in DNA by Pyrosequencing

Ronaghi

Nygren

Lundeberg

et al. 1999

Analytical Biochemistry

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“…), nuclease degradation and denaturing electrophoresis conditions (3)(4)(5). They are stabilized through two G-C base pairs and a non Watson-Crick G-A pair (2,6) ( Figure 1).…”

Section: Hybridization Kinetics Of Oligodeoxyribonucleotides With a Dmentioning

confidence: 99%

Hybridization Kinetics of Oligodeoxyribonucleotides with a d(GCGAAGC) Hairpin at the 3′-End

Chraïbi¹,

Réfrégiers²,

Jollès³

et al. 1999

Journal of Biomolecular Structure and Dynamics

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In order to protect them against enzymatic attack of serum in the antisense strategy, oligodeoxyribonucleotides can be protected on their 3'-side by the sequence d(GCGAAGC) which spontaneously forms a hairpin which is known for its extraordinary stability with regard to thermal denaturation or nuclease degradation (I. Hirao, G. Kawai, S. Yoshizawa, Y. Nishimura, Y. Ishido, K. Watanabe and K. Miura, Nucleic Acids Res. 22, 576-582 (1994)). By contrast, the hairpin does not prevent hybridization of the 5'-stem part of the oligonucleotide to a target DNA strand. As soon as this pairing occurs, the stability of the hairpin is disrupted. Its opening rate, followed by its pairing if possible, is of the same order than that of hybridization of the stem part.

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Nuclease resistance of an extraordinarily thermostable mini-hairpin DNA fragment, d(GCGAAGC) and its application toin vitroprotein synthesis

Cited by 57 publications

References 24 publications

Supercoil-induced extrusion of a regulatory DNA hairpin

Supercoil-induced extrusion of a regulatory DNA hairpin

Analyses of Secondary Structures in DNA by Pyrosequencing

Hybridization Kinetics of Oligodeoxyribonucleotides with a d(GCGAAGC) Hairpin at the 3′-End

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