A-tract and (+)-CC-1065-induced bending of DNA. Comparison of structural features using non-denaturing gel analysis, hydroxyl-radical footprinting, and high-field NMR

Daekyu Sun, Chin Hsiung Lin, Laurence Hurley

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

(+)-CC-1065 is a biologically potent DNA-reactive antitumor antibiotic produced by Streptomyces zelensis. In a previous study we have reported that (+)-CC-1065 produces bending of DNA that has similarities to that intrinsically associated with A-tracts [Lin, C. H., Sun, D., & Hurley, L. H. (1991) Chem. Res. Toxicol. 4, 21-26]. In this article we provide evidence using a combination of non-denaturing gel analysis, hydroxyl-radical footprinting, and high-field NMR for both distinctions between the two types of bends and the importance of junctions in both types of bends. For A-tracts we demonstrate that the locus of bending is at the center of an A-tract and that upon modification of the 3′ adenine with (+)-CC-1065 this locus is moved less than 1 base pair to the 3′ side, and the bending magnitude is significantly increased. For drug bonding sequences such as 5′-AGTTA* or 5′-GATTA* (where * denotes the drug bonding site), the locus of bending is found to be between the two thymines, and the bending is focused over a 2-base-pair sequence rather than a 5-base-pair sequence, as is the case for the A-tract. An important distinction between an A-tract intrinsic bend and a (+)-CC-1065-induced bend is the effect of temperature. While, as shown previously, the magnitude of A-tract bending increases with decrease in temperature, for drug-induced bending of 5′-AGTTA* the bending magnitude increases with increased temperature. Hydroxyl-radical footprinting of the drug-modified 5′-AGTTA* sequence shows a decrease in cleavage centered around the TT sequence, which is presumably associated with a decrease in minor groove width. In a parallel study, the non-self-complementary 12-mer duplex (5′-GGCGGAGTTA*GG-3′)·(5′-CCTAACTC-CGCC-3′) (Figure 2B) and the corresponding (+)-CC-1065-modified duplex adduct were examined thoroughly by one- and two-dimensional 1H NMR and NOESY restrained molecular mechanics and dynamics calculations. Both the 12-mer duplex and the (+)-CC-1065-12-mer duplex adduct maintain an overall B-form DNA with the anti base orientation throughout in aqueous solution at room temperature. The 18C nucleotide of both the 12-mer duplex and its drug-modified adduct has an average C3′-endo sugar pucker The 12-mer duplex exhibits a unique internal motion at the 16A nucleotide, which is located to the 3′ side of the complementary partner of the covalently modified adenine, and a major kink at the 18C-19T step Following covalent bonding with (+)-CC-1065, the discontinuity around 18C is entrapped and further exaggerated. In addition, the 12-mer duplex adduct displays a compression of the minor groove at the 8T to 9T step and widening on both sides, but especially abruptly at the covalent modification site. Structurally the 12-mer duplex adduct bears many similarities to a bent DNA structure, which is intrinsically associated with A-tracts. The major drug-induced distortion on DNA is localized at the 9T and 10A step of the covalently modified strand. A truncated junction model for the drug-entrapped/induced bending of DNA is proposed, and a comparison to intrinsic A-tract bending is made.

Original languageEnglish (US)
Pages (from-to)4487-4495
Number of pages9
JournalBiochemistry
Volume32
Issue number17
StatePublished - 1993
Externally publishedYes

Fingerprint

CC 1065
Hydroxyl Radical
Gels
Nuclear magnetic resonance
DNA
Pharmaceutical Preparations
Base Pairing
Temperature
Adenine
Nucleotides
B-Form DNA
Molecular mechanics
Thymine
Streptomyces
Solar System
Molecular Dynamics Simulation
Mechanics
Sugars
Sun
Molecular dynamics

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{066a5fc8c71043f393bfd0b5d254b5aa,
title = "A-tract and (+)-CC-1065-induced bending of DNA. Comparison of structural features using non-denaturing gel analysis, hydroxyl-radical footprinting, and high-field NMR",
abstract = "(+)-CC-1065 is a biologically potent DNA-reactive antitumor antibiotic produced by Streptomyces zelensis. In a previous study we have reported that (+)-CC-1065 produces bending of DNA that has similarities to that intrinsically associated with A-tracts [Lin, C. H., Sun, D., & Hurley, L. H. (1991) Chem. Res. Toxicol. 4, 21-26]. In this article we provide evidence using a combination of non-denaturing gel analysis, hydroxyl-radical footprinting, and high-field NMR for both distinctions between the two types of bends and the importance of junctions in both types of bends. For A-tracts we demonstrate that the locus of bending is at the center of an A-tract and that upon modification of the 3′ adenine with (+)-CC-1065 this locus is moved less than 1 base pair to the 3′ side, and the bending magnitude is significantly increased. For drug bonding sequences such as 5′-AGTTA* or 5′-GATTA* (where * denotes the drug bonding site), the locus of bending is found to be between the two thymines, and the bending is focused over a 2-base-pair sequence rather than a 5-base-pair sequence, as is the case for the A-tract. An important distinction between an A-tract intrinsic bend and a (+)-CC-1065-induced bend is the effect of temperature. While, as shown previously, the magnitude of A-tract bending increases with decrease in temperature, for drug-induced bending of 5′-AGTTA* the bending magnitude increases with increased temperature. Hydroxyl-radical footprinting of the drug-modified 5′-AGTTA* sequence shows a decrease in cleavage centered around the TT sequence, which is presumably associated with a decrease in minor groove width. In a parallel study, the non-self-complementary 12-mer duplex (5′-GGCGGAGTTA*GG-3′)·(5′-CCTAACTC-CGCC-3′) (Figure 2B) and the corresponding (+)-CC-1065-modified duplex adduct were examined thoroughly by one- and two-dimensional 1H NMR and NOESY restrained molecular mechanics and dynamics calculations. Both the 12-mer duplex and the (+)-CC-1065-12-mer duplex adduct maintain an overall B-form DNA with the anti base orientation throughout in aqueous solution at room temperature. The 18C nucleotide of both the 12-mer duplex and its drug-modified adduct has an average C3′-endo sugar pucker The 12-mer duplex exhibits a unique internal motion at the 16A nucleotide, which is located to the 3′ side of the complementary partner of the covalently modified adenine, and a major kink at the 18C-19T step Following covalent bonding with (+)-CC-1065, the discontinuity around 18C is entrapped and further exaggerated. In addition, the 12-mer duplex adduct displays a compression of the minor groove at the 8T to 9T step and widening on both sides, but especially abruptly at the covalent modification site. Structurally the 12-mer duplex adduct bears many similarities to a bent DNA structure, which is intrinsically associated with A-tracts. The major drug-induced distortion on DNA is localized at the 9T and 10A step of the covalently modified strand. A truncated junction model for the drug-entrapped/induced bending of DNA is proposed, and a comparison to intrinsic A-tract bending is made.",
author = "Daekyu Sun and Lin, {Chin Hsiung} and Laurence Hurley",
year = "1993",
language = "English (US)",
volume = "32",
pages = "4487--4495",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "17",

}

TY - JOUR

T1 - A-tract and (+)-CC-1065-induced bending of DNA. Comparison of structural features using non-denaturing gel analysis, hydroxyl-radical footprinting, and high-field NMR

AU - Sun, Daekyu

AU - Lin, Chin Hsiung

AU - Hurley, Laurence

PY - 1993

Y1 - 1993

N2 - (+)-CC-1065 is a biologically potent DNA-reactive antitumor antibiotic produced by Streptomyces zelensis. In a previous study we have reported that (+)-CC-1065 produces bending of DNA that has similarities to that intrinsically associated with A-tracts [Lin, C. H., Sun, D., & Hurley, L. H. (1991) Chem. Res. Toxicol. 4, 21-26]. In this article we provide evidence using a combination of non-denaturing gel analysis, hydroxyl-radical footprinting, and high-field NMR for both distinctions between the two types of bends and the importance of junctions in both types of bends. For A-tracts we demonstrate that the locus of bending is at the center of an A-tract and that upon modification of the 3′ adenine with (+)-CC-1065 this locus is moved less than 1 base pair to the 3′ side, and the bending magnitude is significantly increased. For drug bonding sequences such as 5′-AGTTA* or 5′-GATTA* (where * denotes the drug bonding site), the locus of bending is found to be between the two thymines, and the bending is focused over a 2-base-pair sequence rather than a 5-base-pair sequence, as is the case for the A-tract. An important distinction between an A-tract intrinsic bend and a (+)-CC-1065-induced bend is the effect of temperature. While, as shown previously, the magnitude of A-tract bending increases with decrease in temperature, for drug-induced bending of 5′-AGTTA* the bending magnitude increases with increased temperature. Hydroxyl-radical footprinting of the drug-modified 5′-AGTTA* sequence shows a decrease in cleavage centered around the TT sequence, which is presumably associated with a decrease in minor groove width. In a parallel study, the non-self-complementary 12-mer duplex (5′-GGCGGAGTTA*GG-3′)·(5′-CCTAACTC-CGCC-3′) (Figure 2B) and the corresponding (+)-CC-1065-modified duplex adduct were examined thoroughly by one- and two-dimensional 1H NMR and NOESY restrained molecular mechanics and dynamics calculations. Both the 12-mer duplex and the (+)-CC-1065-12-mer duplex adduct maintain an overall B-form DNA with the anti base orientation throughout in aqueous solution at room temperature. The 18C nucleotide of both the 12-mer duplex and its drug-modified adduct has an average C3′-endo sugar pucker The 12-mer duplex exhibits a unique internal motion at the 16A nucleotide, which is located to the 3′ side of the complementary partner of the covalently modified adenine, and a major kink at the 18C-19T step Following covalent bonding with (+)-CC-1065, the discontinuity around 18C is entrapped and further exaggerated. In addition, the 12-mer duplex adduct displays a compression of the minor groove at the 8T to 9T step and widening on both sides, but especially abruptly at the covalent modification site. Structurally the 12-mer duplex adduct bears many similarities to a bent DNA structure, which is intrinsically associated with A-tracts. The major drug-induced distortion on DNA is localized at the 9T and 10A step of the covalently modified strand. A truncated junction model for the drug-entrapped/induced bending of DNA is proposed, and a comparison to intrinsic A-tract bending is made.

AB - (+)-CC-1065 is a biologically potent DNA-reactive antitumor antibiotic produced by Streptomyces zelensis. In a previous study we have reported that (+)-CC-1065 produces bending of DNA that has similarities to that intrinsically associated with A-tracts [Lin, C. H., Sun, D., & Hurley, L. H. (1991) Chem. Res. Toxicol. 4, 21-26]. In this article we provide evidence using a combination of non-denaturing gel analysis, hydroxyl-radical footprinting, and high-field NMR for both distinctions between the two types of bends and the importance of junctions in both types of bends. For A-tracts we demonstrate that the locus of bending is at the center of an A-tract and that upon modification of the 3′ adenine with (+)-CC-1065 this locus is moved less than 1 base pair to the 3′ side, and the bending magnitude is significantly increased. For drug bonding sequences such as 5′-AGTTA* or 5′-GATTA* (where * denotes the drug bonding site), the locus of bending is found to be between the two thymines, and the bending is focused over a 2-base-pair sequence rather than a 5-base-pair sequence, as is the case for the A-tract. An important distinction between an A-tract intrinsic bend and a (+)-CC-1065-induced bend is the effect of temperature. While, as shown previously, the magnitude of A-tract bending increases with decrease in temperature, for drug-induced bending of 5′-AGTTA* the bending magnitude increases with increased temperature. Hydroxyl-radical footprinting of the drug-modified 5′-AGTTA* sequence shows a decrease in cleavage centered around the TT sequence, which is presumably associated with a decrease in minor groove width. In a parallel study, the non-self-complementary 12-mer duplex (5′-GGCGGAGTTA*GG-3′)·(5′-CCTAACTC-CGCC-3′) (Figure 2B) and the corresponding (+)-CC-1065-modified duplex adduct were examined thoroughly by one- and two-dimensional 1H NMR and NOESY restrained molecular mechanics and dynamics calculations. Both the 12-mer duplex and the (+)-CC-1065-12-mer duplex adduct maintain an overall B-form DNA with the anti base orientation throughout in aqueous solution at room temperature. The 18C nucleotide of both the 12-mer duplex and its drug-modified adduct has an average C3′-endo sugar pucker The 12-mer duplex exhibits a unique internal motion at the 16A nucleotide, which is located to the 3′ side of the complementary partner of the covalently modified adenine, and a major kink at the 18C-19T step Following covalent bonding with (+)-CC-1065, the discontinuity around 18C is entrapped and further exaggerated. In addition, the 12-mer duplex adduct displays a compression of the minor groove at the 8T to 9T step and widening on both sides, but especially abruptly at the covalent modification site. Structurally the 12-mer duplex adduct bears many similarities to a bent DNA structure, which is intrinsically associated with A-tracts. The major drug-induced distortion on DNA is localized at the 9T and 10A step of the covalently modified strand. A truncated junction model for the drug-entrapped/induced bending of DNA is proposed, and a comparison to intrinsic A-tract bending is made.

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