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Kinking the double helix by bending deformation.

Du Q, Kotlyar A, Vologodskii A - Nucleic Acids Res. (2007)

Bottom Line: To detect structural disruptions in the minicircles we treated them by single-strand-specific endonucleases.The data showed that the regular DNA structure is disrupted by bending deformation in the 64-65-bp minicircles, but not in the 85-86-bp minicircles.Our results suggest that strong DNA bending initiates kink formation while preserving base pairing.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, New York University, New York, NY 10003, USA.

ABSTRACT
DNA bending and torsional deformations that often occur during its functioning inside the cell can cause local disruptions of the regular helical structure. The disruptions created by negative torsional stress have been studied in detail, but those caused by bending stress have only been analyzed theoretically. By probing the structure of very small DNA circles, we determined that bending stress disrupts the regular helical structure when the radius of DNA curvature is smaller than 3.5 nm. First, we developed an efficient method to obtain covalently closed DNA minicircles. To detect structural disruptions in the minicircles we treated them by single-strand-specific endonucleases. The data showed that the regular DNA structure is disrupted by bending deformation in the 64-65-bp minicircles, but not in the 85-86-bp minicircles. Our results suggest that strong DNA bending initiates kink formation while preserving base pairing.

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Probing the structure of 100 bp and 106 bp DNA minicircles by single-strand-specific endonucleases. The denaturing polyacrylamide gels show the results of 60 min treatment by BAL 31 (a) and 30-min treatment by S1 (b). The minicircles (ds circular) of 100 bp are negatively supercoiled (), while minicircles of 106 bp are relaxed. The other labels refer to single-stranded linear fragments (ss linear) and circles (ss circular). The minicircle size is shown at the bottom of the figure.
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Figure 5: Probing the structure of 100 bp and 106 bp DNA minicircles by single-strand-specific endonucleases. The denaturing polyacrylamide gels show the results of 60 min treatment by BAL 31 (a) and 30-min treatment by S1 (b). The minicircles (ds circular) of 100 bp are negatively supercoiled (), while minicircles of 106 bp are relaxed. The other labels refer to single-stranded linear fragments (ss linear) and circles (ss circular). The minicircle size is shown at the bottom of the figure.

Mentions: Second, we studied the nuclease digestion of 106 and 100 bp minicircles. The data in Figure 5 show that the DNA structure is not disrupted in the 106 bp minicircles that are represented by the single torsionally unstressed topoisomer (ΔLk = 0) (17). The fragment of 100 bp has a semi-integer number of helix turns, so one could expect that its cyclization gives two topoisomers. The minicircles, however, are presented by only one topoisomer that is quickly digested by BAL 31 (Figure 5a). Thus, the topoisomer has well-pronounced local disruptions. It means that its ΔLk equals –0.5 rather than +0.5, since only negative torsional stress can promote the disruptions [see Equation (3)]. Correspondingly, the free energy of the (+) topoisomer is higher than that of (–) topoisomer, and (+) topoisomer does not appear during the ligation. S1 endonuclease also digests the topoisomer, although the reaction proceeds very slowly (Figure 5b).Figure 5.


Kinking the double helix by bending deformation.

Du Q, Kotlyar A, Vologodskii A - Nucleic Acids Res. (2007)

Probing the structure of 100 bp and 106 bp DNA minicircles by single-strand-specific endonucleases. The denaturing polyacrylamide gels show the results of 60 min treatment by BAL 31 (a) and 30-min treatment by S1 (b). The minicircles (ds circular) of 100 bp are negatively supercoiled (), while minicircles of 106 bp are relaxed. The other labels refer to single-stranded linear fragments (ss linear) and circles (ss circular). The minicircle size is shown at the bottom of the figure.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2275110&req=5

Figure 5: Probing the structure of 100 bp and 106 bp DNA minicircles by single-strand-specific endonucleases. The denaturing polyacrylamide gels show the results of 60 min treatment by BAL 31 (a) and 30-min treatment by S1 (b). The minicircles (ds circular) of 100 bp are negatively supercoiled (), while minicircles of 106 bp are relaxed. The other labels refer to single-stranded linear fragments (ss linear) and circles (ss circular). The minicircle size is shown at the bottom of the figure.
Mentions: Second, we studied the nuclease digestion of 106 and 100 bp minicircles. The data in Figure 5 show that the DNA structure is not disrupted in the 106 bp minicircles that are represented by the single torsionally unstressed topoisomer (ΔLk = 0) (17). The fragment of 100 bp has a semi-integer number of helix turns, so one could expect that its cyclization gives two topoisomers. The minicircles, however, are presented by only one topoisomer that is quickly digested by BAL 31 (Figure 5a). Thus, the topoisomer has well-pronounced local disruptions. It means that its ΔLk equals –0.5 rather than +0.5, since only negative torsional stress can promote the disruptions [see Equation (3)]. Correspondingly, the free energy of the (+) topoisomer is higher than that of (–) topoisomer, and (+) topoisomer does not appear during the ligation. S1 endonuclease also digests the topoisomer, although the reaction proceeds very slowly (Figure 5b).Figure 5.

Bottom Line: To detect structural disruptions in the minicircles we treated them by single-strand-specific endonucleases.The data showed that the regular DNA structure is disrupted by bending deformation in the 64-65-bp minicircles, but not in the 85-86-bp minicircles.Our results suggest that strong DNA bending initiates kink formation while preserving base pairing.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, New York University, New York, NY 10003, USA.

ABSTRACT
DNA bending and torsional deformations that often occur during its functioning inside the cell can cause local disruptions of the regular helical structure. The disruptions created by negative torsional stress have been studied in detail, but those caused by bending stress have only been analyzed theoretically. By probing the structure of very small DNA circles, we determined that bending stress disrupts the regular helical structure when the radius of DNA curvature is smaller than 3.5 nm. First, we developed an efficient method to obtain covalently closed DNA minicircles. To detect structural disruptions in the minicircles we treated them by single-strand-specific endonucleases. The data showed that the regular DNA structure is disrupted by bending deformation in the 64-65-bp minicircles, but not in the 85-86-bp minicircles. Our results suggest that strong DNA bending initiates kink formation while preserving base pairing.

Show MeSH