In vitro ligation of oligodeoxynucleotides containing C8-oxidized purine lesions using bacteriophage T4 DNA ligase

Xiaobei Zhao, James G. Muller, Mohan Halasyam, Sheila S. David, Cynthia J. Burrows

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Ligases conduct the final stage of repair of DNA damage by sealing a single-stranded nick after excision of damaged nucleotides and reinsertion of correct nucleotides. Depending upon the circumstances and the success of the repair process, lesions may remain at the ligation site, either in the template or at the oligomer termini to be joined. Ligation experiments using bacteriophage T4 DNA ligase were carried out with purine lesions in four positions surrounding the nick site in a total of 96 different duplexes. The oxidized lesion 8-oxo-7,8-dihydroguanosine (OG) showed, as expected, that the enzyme is most sensitive to lesions on the 3′ end of the nick compared to the 5′ end and to lesions located in the intact template strand. In general, substrates containing the OG·A mismatch were more readily ligated than those with the OG·C mismatch. Ligations of duplexes containing the OA·T base pair (OA = 8-oxo-7,8-dihydroadenosine) that could adopt an anti-anti conformation proceeded with high efficiencies. An OI·A mismatch-containing duplex (OI = 8-oxo-7,8-dihydroinosine) behaved like OG·A. Due to its low reduction potential, OG is readily oxidized to secondary oxidation products, such as the guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) nucleosides; these lesions also contain an oxo group at the original C8 position of the purine. Ligation of oligomers containing Gh and Sp occurred when opposite A and G, although the overall ligation efficiencies were much lower than those of most OG base pairs. Steady-state kinetic studies were carried out for representative examples of lesions in the template. Km increased by 90-100-fold for OG·C-, OI·C-, OI·A-, and OA·T-containing duplexes compared to that of a G·C-containing duplex. Substrates containing Gh·A, Gh·G, Sp·A, and Sp·G base pairs exhibited Km values 20-70-fold higher than that of the substrate containing a G·C base pair, while the Km value for OG·A was 5 times lower than that for G·C.

Original languageEnglish (US)
Pages (from-to)3734-3744
Number of pages11
JournalBiochemistry
Volume46
Issue number12
DOIs
StatePublished - Mar 27 2007
Externally publishedYes

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DNA Ligases
Bacteriophage T4
Bacteriophages
Oligodeoxyribonucleotides
Ligation
Base Pairing
Oligomers
Repair
Substrates
Nucleotides
Ligases
Conformations
Oxidation
Kinetics
DNA
DNA Damage
Enzymes
purine
In Vitro Techniques
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

In vitro ligation of oligodeoxynucleotides containing C8-oxidized purine lesions using bacteriophage T4 DNA ligase. / Zhao, Xiaobei; Muller, James G.; Halasyam, Mohan; David, Sheila S.; Burrows, Cynthia J.

In: Biochemistry, Vol. 46, No. 12, 27.03.2007, p. 3734-3744.

Research output: Contribution to journalArticle

Zhao, Xiaobei ; Muller, James G. ; Halasyam, Mohan ; David, Sheila S. ; Burrows, Cynthia J. / In vitro ligation of oligodeoxynucleotides containing C8-oxidized purine lesions using bacteriophage T4 DNA ligase. In: Biochemistry. 2007 ; Vol. 46, No. 12. pp. 3734-3744.
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abstract = "Ligases conduct the final stage of repair of DNA damage by sealing a single-stranded nick after excision of damaged nucleotides and reinsertion of correct nucleotides. Depending upon the circumstances and the success of the repair process, lesions may remain at the ligation site, either in the template or at the oligomer termini to be joined. Ligation experiments using bacteriophage T4 DNA ligase were carried out with purine lesions in four positions surrounding the nick site in a total of 96 different duplexes. The oxidized lesion 8-oxo-7,8-dihydroguanosine (OG) showed, as expected, that the enzyme is most sensitive to lesions on the 3′ end of the nick compared to the 5′ end and to lesions located in the intact template strand. In general, substrates containing the OG·A mismatch were more readily ligated than those with the OG·C mismatch. Ligations of duplexes containing the OA·T base pair (OA = 8-oxo-7,8-dihydroadenosine) that could adopt an anti-anti conformation proceeded with high efficiencies. An OI·A mismatch-containing duplex (OI = 8-oxo-7,8-dihydroinosine) behaved like OG·A. Due to its low reduction potential, OG is readily oxidized to secondary oxidation products, such as the guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) nucleosides; these lesions also contain an oxo group at the original C8 position of the purine. Ligation of oligomers containing Gh and Sp occurred when opposite A and G, although the overall ligation efficiencies were much lower than those of most OG base pairs. Steady-state kinetic studies were carried out for representative examples of lesions in the template. Km increased by 90-100-fold for OG·C-, OI·C-, OI·A-, and OA·T-containing duplexes compared to that of a G·C-containing duplex. Substrates containing Gh·A, Gh·G, Sp·A, and Sp·G base pairs exhibited Km values 20-70-fold higher than that of the substrate containing a G·C base pair, while the Km value for OG·A was 5 times lower than that for G·C.",
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AU - Zhao, Xiaobei

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AU - Burrows, Cynthia J.

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AB - Ligases conduct the final stage of repair of DNA damage by sealing a single-stranded nick after excision of damaged nucleotides and reinsertion of correct nucleotides. Depending upon the circumstances and the success of the repair process, lesions may remain at the ligation site, either in the template or at the oligomer termini to be joined. Ligation experiments using bacteriophage T4 DNA ligase were carried out with purine lesions in four positions surrounding the nick site in a total of 96 different duplexes. The oxidized lesion 8-oxo-7,8-dihydroguanosine (OG) showed, as expected, that the enzyme is most sensitive to lesions on the 3′ end of the nick compared to the 5′ end and to lesions located in the intact template strand. In general, substrates containing the OG·A mismatch were more readily ligated than those with the OG·C mismatch. Ligations of duplexes containing the OA·T base pair (OA = 8-oxo-7,8-dihydroadenosine) that could adopt an anti-anti conformation proceeded with high efficiencies. An OI·A mismatch-containing duplex (OI = 8-oxo-7,8-dihydroinosine) behaved like OG·A. Due to its low reduction potential, OG is readily oxidized to secondary oxidation products, such as the guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) nucleosides; these lesions also contain an oxo group at the original C8 position of the purine. Ligation of oligomers containing Gh and Sp occurred when opposite A and G, although the overall ligation efficiencies were much lower than those of most OG base pairs. Steady-state kinetic studies were carried out for representative examples of lesions in the template. Km increased by 90-100-fold for OG·C-, OI·C-, OI·A-, and OA·T-containing duplexes compared to that of a G·C-containing duplex. Substrates containing Gh·A, Gh·G, Sp·A, and Sp·G base pairs exhibited Km values 20-70-fold higher than that of the substrate containing a G·C base pair, while the Km value for OG·A was 5 times lower than that for G·C.

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