Opposite stereoselective resistance to digestion by phosphodiesterases I and II of benzo[a]pyrene diol epoxide-modified oligonucleotide adducts

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Abstract

The deoxyribooligonucleotide 5′-d(CTCACATGTACACTCT) was reacted separately with the chiral diol epoxide isomers 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE)] and 7α,8β-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-anti-BPDE)], to produce the modified oligonucleotides 5′-d(CTCACATGBPDETACACTCT). Adducts in which either (+)-anti-BPDE or (-)-anti-BPDE are covalently bound via their C10 positions by trans addition to the exocyclic amino group of the single G residues were isolated and purified by HPLC methods. Snake venom phosphodiesterase (SVPD, phosphodiesterase I), which hydrolyzes DNA from the 3′-OH terminus to the 5′-end, digests the (+)-trans-anti-BPDE-oligonucleotide adducts at a significantly faster rate than that of the sterically different (-)-trans-anti-BPDE-oligonucleotide adducts. However, using spleen phosphodiesterase (SPD, phosphodiesterase II), which hydrolyzes DNA in the 5′ → 3′ direction, the opposite stereoselective resistance to digestion is observed. Using shorter BPDE-modified oligonucleotides as standards, the enzyme stall sites have been defined by gel electrophoresis methods; the most digestion-resistant phosphodiester linkage is the 5′-d(...T-G*...)-3′ bond in the case of (+)-trans-BPDE-modified oligonucleotide adducts for both enzymes, SVPD and SPD (the starred G denotes the site of BPDE modification). In the case of the (-)-trans-BPDE-modified oligonucleotide adducts, the phosphodiester bond on the 3′-side of the modified G [5′-d(...G*-T...)-3′] is most resistant to digestion by both enzymes. It is concluded that in single-stranded oligonucleotides the pyrenyl residues point toward the 5′-end, while in (-)-trans adducts they point toward the 3′-end of the modified strands, paralleling the orientations recently found by 2D NMR techniques in analogous (+)-trans- and (-)-trans-BPDE-oligonucleotide duplexes [de los Santos et al. (1992) Biochemistry 31, 5245-5252]. Overall rates of enzyme digestion are more hindered whenever the bulky pyrenyl residue points in a direction opposing the progress of hydrolysis and are more facile when the pyrenyl ring points along the direction (3′ → 5′ or 5′ → 3′) of exonuclease digestion. Identification of exonuclease digestion stall sites in single-stranded oligonucleotides modified with bulky adducts may prove useful for deducing the orientations of these covalently bound residues relative to the modified base and the 5′-3′ strand polarity.

Original languageEnglish (US)
Pages (from-to)11785-11793
Number of pages9
JournalBiochemistry
Volume32
Issue number44
StatePublished - 1993

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spleen exonuclease
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide
Phosphodiesterase I
Benzo(a)pyrene
Epoxy Compounds
Oligonucleotides
Digestion
Exonucleases
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{281ece7cb86048eaa8c63ed729d3bc3b,
title = "Opposite stereoselective resistance to digestion by phosphodiesterases I and II of benzo[a]pyrene diol epoxide-modified oligonucleotide adducts",
abstract = "The deoxyribooligonucleotide 5′-d(CTCACATGTACACTCT) was reacted separately with the chiral diol epoxide isomers 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE)] and 7α,8β-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-anti-BPDE)], to produce the modified oligonucleotides 5′-d(CTCACATGBPDETACACTCT). Adducts in which either (+)-anti-BPDE or (-)-anti-BPDE are covalently bound via their C10 positions by trans addition to the exocyclic amino group of the single G residues were isolated and purified by HPLC methods. Snake venom phosphodiesterase (SVPD, phosphodiesterase I), which hydrolyzes DNA from the 3′-OH terminus to the 5′-end, digests the (+)-trans-anti-BPDE-oligonucleotide adducts at a significantly faster rate than that of the sterically different (-)-trans-anti-BPDE-oligonucleotide adducts. However, using spleen phosphodiesterase (SPD, phosphodiesterase II), which hydrolyzes DNA in the 5′ → 3′ direction, the opposite stereoselective resistance to digestion is observed. Using shorter BPDE-modified oligonucleotides as standards, the enzyme stall sites have been defined by gel electrophoresis methods; the most digestion-resistant phosphodiester linkage is the 5′-d(...T-G*...)-3′ bond in the case of (+)-trans-BPDE-modified oligonucleotide adducts for both enzymes, SVPD and SPD (the starred G denotes the site of BPDE modification). In the case of the (-)-trans-BPDE-modified oligonucleotide adducts, the phosphodiester bond on the 3′-side of the modified G [5′-d(...G*-T...)-3′] is most resistant to digestion by both enzymes. It is concluded that in single-stranded oligonucleotides the pyrenyl residues point toward the 5′-end, while in (-)-trans adducts they point toward the 3′-end of the modified strands, paralleling the orientations recently found by 2D NMR techniques in analogous (+)-trans- and (-)-trans-BPDE-oligonucleotide duplexes [de los Santos et al. (1992) Biochemistry 31, 5245-5252]. Overall rates of enzyme digestion are more hindered whenever the bulky pyrenyl residue points in a direction opposing the progress of hydrolysis and are more facile when the pyrenyl ring points along the direction (3′ → 5′ or 5′ → 3′) of exonuclease digestion. Identification of exonuclease digestion stall sites in single-stranded oligonucleotides modified with bulky adducts may prove useful for deducing the orientations of these covalently bound residues relative to the modified base and the 5′-3′ strand polarity.",
author = "Nicholas Geacintov",
year = "1993",
language = "English (US)",
volume = "32",
pages = "11785--11793",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "44",

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TY - JOUR

T1 - Opposite stereoselective resistance to digestion by phosphodiesterases I and II of benzo[a]pyrene diol epoxide-modified oligonucleotide adducts

AU - Geacintov, Nicholas

PY - 1993

Y1 - 1993

N2 - The deoxyribooligonucleotide 5′-d(CTCACATGTACACTCT) was reacted separately with the chiral diol epoxide isomers 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE)] and 7α,8β-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-anti-BPDE)], to produce the modified oligonucleotides 5′-d(CTCACATGBPDETACACTCT). Adducts in which either (+)-anti-BPDE or (-)-anti-BPDE are covalently bound via their C10 positions by trans addition to the exocyclic amino group of the single G residues were isolated and purified by HPLC methods. Snake venom phosphodiesterase (SVPD, phosphodiesterase I), which hydrolyzes DNA from the 3′-OH terminus to the 5′-end, digests the (+)-trans-anti-BPDE-oligonucleotide adducts at a significantly faster rate than that of the sterically different (-)-trans-anti-BPDE-oligonucleotide adducts. However, using spleen phosphodiesterase (SPD, phosphodiesterase II), which hydrolyzes DNA in the 5′ → 3′ direction, the opposite stereoselective resistance to digestion is observed. Using shorter BPDE-modified oligonucleotides as standards, the enzyme stall sites have been defined by gel electrophoresis methods; the most digestion-resistant phosphodiester linkage is the 5′-d(...T-G*...)-3′ bond in the case of (+)-trans-BPDE-modified oligonucleotide adducts for both enzymes, SVPD and SPD (the starred G denotes the site of BPDE modification). In the case of the (-)-trans-BPDE-modified oligonucleotide adducts, the phosphodiester bond on the 3′-side of the modified G [5′-d(...G*-T...)-3′] is most resistant to digestion by both enzymes. It is concluded that in single-stranded oligonucleotides the pyrenyl residues point toward the 5′-end, while in (-)-trans adducts they point toward the 3′-end of the modified strands, paralleling the orientations recently found by 2D NMR techniques in analogous (+)-trans- and (-)-trans-BPDE-oligonucleotide duplexes [de los Santos et al. (1992) Biochemistry 31, 5245-5252]. Overall rates of enzyme digestion are more hindered whenever the bulky pyrenyl residue points in a direction opposing the progress of hydrolysis and are more facile when the pyrenyl ring points along the direction (3′ → 5′ or 5′ → 3′) of exonuclease digestion. Identification of exonuclease digestion stall sites in single-stranded oligonucleotides modified with bulky adducts may prove useful for deducing the orientations of these covalently bound residues relative to the modified base and the 5′-3′ strand polarity.

AB - The deoxyribooligonucleotide 5′-d(CTCACATGTACACTCT) was reacted separately with the chiral diol epoxide isomers 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE)] and 7α,8β-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-anti-BPDE)], to produce the modified oligonucleotides 5′-d(CTCACATGBPDETACACTCT). Adducts in which either (+)-anti-BPDE or (-)-anti-BPDE are covalently bound via their C10 positions by trans addition to the exocyclic amino group of the single G residues were isolated and purified by HPLC methods. Snake venom phosphodiesterase (SVPD, phosphodiesterase I), which hydrolyzes DNA from the 3′-OH terminus to the 5′-end, digests the (+)-trans-anti-BPDE-oligonucleotide adducts at a significantly faster rate than that of the sterically different (-)-trans-anti-BPDE-oligonucleotide adducts. However, using spleen phosphodiesterase (SPD, phosphodiesterase II), which hydrolyzes DNA in the 5′ → 3′ direction, the opposite stereoselective resistance to digestion is observed. Using shorter BPDE-modified oligonucleotides as standards, the enzyme stall sites have been defined by gel electrophoresis methods; the most digestion-resistant phosphodiester linkage is the 5′-d(...T-G*...)-3′ bond in the case of (+)-trans-BPDE-modified oligonucleotide adducts for both enzymes, SVPD and SPD (the starred G denotes the site of BPDE modification). In the case of the (-)-trans-BPDE-modified oligonucleotide adducts, the phosphodiester bond on the 3′-side of the modified G [5′-d(...G*-T...)-3′] is most resistant to digestion by both enzymes. It is concluded that in single-stranded oligonucleotides the pyrenyl residues point toward the 5′-end, while in (-)-trans adducts they point toward the 3′-end of the modified strands, paralleling the orientations recently found by 2D NMR techniques in analogous (+)-trans- and (-)-trans-BPDE-oligonucleotide duplexes [de los Santos et al. (1992) Biochemistry 31, 5245-5252]. Overall rates of enzyme digestion are more hindered whenever the bulky pyrenyl residue points in a direction opposing the progress of hydrolysis and are more facile when the pyrenyl ring points along the direction (3′ → 5′ or 5′ → 3′) of exonuclease digestion. Identification of exonuclease digestion stall sites in single-stranded oligonucleotides modified with bulky adducts may prove useful for deducing the orientations of these covalently bound residues relative to the modified base and the 5′-3′ strand polarity.

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