Conformational and thermodynamic consequences of the introduction of a nick in duplexed DNA fragments: an NMR study augmented by biochemical experiments

Pieters, Jane M. L.; Mans, R. M. W.; Elst, Hans van den; Marel, Gijs A. van den; Boom, Jacques H. van; Altona, C.

doi:10.1093/nar/17.12.4551

Cited by 45 publications

(47 citation statements)

References 24 publications

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“…One of the main conclusions from previous investigations is that the nick barely changes the B DNA configuration and its bending flexibility, at least at high-salt conditions (15)(16)(17)(18)(19). We find that in addition to Ͻ7% increases in bending flexibility for both nicks, the nicks significantly enhance the torsional flexibility, with average twisting fluctuations of 30°at the nick NwP and 64°at the nick N, respectively.…”

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confidence: 46%

High-throughput approach for detection of DNA bending and flexibility based on cyclization

Zhang

Crothers

2003

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

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We have developed a high-throughput approach to the laborintensive problems of DNA cyclization, which we use to characterize DNA curvature and mechanical properties. The method includes a combinatorial approach to make the DNA constructs needed and automated real-time measurement of the kinetics using fluorescence. We validated the approach and investigated the flexibility of two kinds of nicked DNA and AT dinucleotide repeats. We found that, although the nicks hardly alter the bending flexibility, they significantly increase the torsional flexibility, and that the AT repeat has 28% (؎12%) lower bending rigidity than a generic DNA sequence.S equence-dependent DNA curvature and flexibility are important characteristics of DNA structure (1-5), which influences almost every aspect of DNA-related processes (6, 7). The idea that DNA sequence information might also be encoded through its 3D shape and mechanical properties may provide a new dimension for functional genomics (8). However, our limited power of mapping DNA tertiary structure from its sequence impedes the testing of this idea, due to lack of a reliable and convenient approach to quantify DNA bending and flexibility (9).Among a variety of approaches that have been used to measure the global curvature and flexibility of DNA, DNA cyclization has the advantage of high sensitivity, a complete theoretical basis, and the capacity to measure almost all interesting features of global geometric and mechanical properties of DNA, including the magnitude and direction of its curvature, helical repeat, and bending and twisting flexibility (10-13). Its principle is illustrated in Fig. 7, which is published as supporting information on the PNAS web site, www.pnas.org. In this method, the J factors of a series of DNA molecules containing the same test sequence are measured and analyzed by using a model that relates them to DNA properties. The J factor is the effective concentration of one end of a DNA molecule at the other and equal to the ratio of the unimolecular ligation rate constant (k 1 ) to the bimolecular ligation rate constant (k 2 ) catalyzed by DNA ligase under certain conditions. Because k 2 is the same for all constructs with the same cohesive ends, the main aim of DNA cyclization is to measure k 1 .The traditional implementation (10, 14) of cyclization involves radioactive labeling of DNA constructs and separation of the ligation mixture by PAGE to obtain data on ligation kinetics. The data are mainly interpreted by Monte Carlo simulation to determine curvature and flexibility parameters. Several aspects limit throughput, including the time needed to prepare the Ϸ15 constructs required for the study of a single sequence. The need for multiple oligonucleotide syntheses, constant optimization of PCR reactions, and the limited lifetime of radioactive labeled constructs make the sample preparation extremely tedious. Second, determining ligation rates from gels is labor-intensive. Finally, data interpretation using Monte Carlo simulation is cumbersome and computat...

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confidence: 46%

High-throughput approach for detection of DNA bending and flexibility based on cyclization

Zhang

Crothers

2003

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

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“…Both prenicking (14,15) and depurination (16,18) are known to destabilize DNA double-helical structure, and prenicking may also increase DNA structural flexibility (19). We found that both defects at positions around the Ϫ10 region had strong stimulatory effect on the rate of open complex formation on the prmup-1⌬265 promoter.…”

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confidence: 73%

Stimulation of Open Complex Formation by Nicks and Apurinic Sites Suggests a Role for Nucleation of DNA Melting in Escherichia coli Promoter Function

McClure

1998

Journal of Biological Chemistry

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We report the effects of depurination and prenicking at various positions of the phage prmup-1⌬265 promoter DNA on the rate of open complex formation. We have found that depurination and prenicking at positions around the ؊10 region strongly stimulated the rate of open complex formation. Since nicking and depurination are known to destabilize DNA helical structure, our observations indicate that the instability of the ؊10 region is important for open complex formation. We further infer that (i) the nucleation of DNA melting, which occurs during the isomerization from the closed complex into the open complex, contributes to the rate of open complex formation; (ii) the nucleation of melting occurs around the ؊10 region; and (iii) the propagation of DNA melting from the nucleation region is not ratelimiting. In addition, we have found that depurination at several positions inhibited open complex formation. We used dimethyl sulfate modification protection studies to show that most of the guanine bases that are among these positions are in contact with RNA polymerase in the open complex.Open complex formation is the major rate-determining step in the process of transcription initiation with E. coli RNA polymerase (E 70 ). Many of the previous studies have been based on a model containing two functional steps (1, 2) that include the initial binding of RNA polymerase to the promoter DNA to form the closed complex followed by the isomerization of the closed complex into the open complex. Some studies have also suggested that the isomerization step in the two-step model can be further dissected into two discrete steps: the rate-limiting isomerization step per se and a DNA melting step (3-10). In this extended model, the DNA melting step is argued to be very rapid under normal transcription conditions, and consequently it is normally not ratelimiting. It has been suggested that the isomerization step involves a major conformational change in RNA polymerase, which is thought to be the cause for the nucleation of DNA melting (10, 11).It is known that the Ϫ10 region of the promoters recognized by 70 holoenzyme is thermodynamically less stable than average DNA. The conserved Ϫ10 hexamer sequence has a melting free energy that is close to the maximum (least stable) possible based on a calculation using the nearest-neighbor thermodynamic data of Breslauer et al. (12). Therefore, as expected, Margalit et al. (13) have shown that 80% of the up and down mutations in the Ϫ10 region correlated qualitatively with the change in the melting free energy. A closer inspection showed that most of the mutations among the exceptions are located outside the Ϫ10 (hexamer) region, and consequently are not bona fide exceptions if only the melting free energy of the Ϫ10 region is important. There were several exceptions within the Ϫ10 region. However, in such cases it can be argued that the effect of the mutation on the specific contact between RNA polymerase and promoter DNA is larger than the effect of the melting free energy change, and con...

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“…On the experimental side, Protozanova et al have measured free energy of stacking for base-pair steps in a nicked DNA duplex (Protozanova et al 2004). It is attractive to generalise these stacking-free energies for nicked DNA to the intact DNA double helix; however, NMR analyses have shown that the broken phosphodiester bond pushes the conformation away from canonical B-form (Pieters et al 1989;Roll et al 1998; Snowden-Ifft & Wemmer, 1990) which leaves . Overall then, while there is significant disagreement on the magnitude (and sometimes the ranking) of the 10 stacking energies for complementary base-pair steps, the key feature of a weak CG stack which points to the existence of a GNC triplet code is preserved.…”

Section: −1mentioning

confidence: 99%

DNA partitions into triplets under tension in the presence of organic cations, with sequence evolutionary age predicting the stability of the triplet phase

Taghavi

Schoot

Berryman

2017

Quart. Rev. Biophys.

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Abstract. Using atomistic simulations, we show the formation of stable triplet structure when particular GC-rich DNA duplexes are extended in solution over a timescale of hundreds of nanoseconds, in the presence of organic salt. We present planar-stacked triplet disproportionated DNA (Σ DNA) as a possible solution phase of the double helix under tension, subject to sequence and the presence of stabilising co-factors. Considering the partitioning of the duplexes into triplets of base pairs as the first step of operation of recombinase enzymes like RecA, we emphasise the structure-function relationship in Σ DNA. We supplement atomistic calculations with thermodynamic arguments to show that codons for 'phase 1' amino acids (those appearing early in evolution) are more likely than a lower entropy GC-rich sequence to form triplets under tension. We further observe that the four amino acids supposed (in the 'GADV world' hypothesis) to constitute the minimal set to produce functional globular proteins have the strongest triplet-forming propensity within the phase 1 set, showing a series of decreasing triplet propensity with evolutionary newness. The weak form of our observation provides a physical mechanism to minimise read frame and recombination alignment errors in the early evolution of the genetic code.

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Conformational and thermodynamic consequences of the introduction of a nick in duplexed DNA fragments: an NMR study augmented by biochemical experiments

Cited by 45 publications

References 24 publications

High-throughput approach for detection of DNA bending and flexibility based on cyclization

High-throughput approach for detection of DNA bending and flexibility based on cyclization

Stimulation of Open Complex Formation by Nicks and Apurinic Sites Suggests a Role for Nucleation of DNA Melting in Escherichia coli Promoter Function

DNA partitions into triplets under tension in the presence of organic cations, with sequence evolutionary age predicting the stability of the triplet phase

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