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Repair of O6-methylguanine adducts in human telomeric G-quadruplex DNA by O6-alkylguanine-DNA alkyltransferase.

Hellman LM, Spear TJ, Koontz CJ, Melikishvili M, Fried MG - Nucleic Acids Res. (2014)

Bottom Line: Its functions with short single-stranded and duplex substrates have been characterized, but its ability to act on other DNA structures remains poorly understood.Here, we examine the functions of this enzyme on O(6)-methylguanine (6mG) adducts in the four-stranded structure of the human telomeric G-quadruplex.This distinction may reflect differences in the conformational dynamics of 6mG residues in G-quadruplex DNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, Lexington, KY 40536, USA.

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Time course of alkyltransfer in G-quadruplex DNAs. (A) Reaction substrate and product detected by electrophoresis. AGT (4 μM) was mixed with G1 DNA (0.25 μM) to start the reaction and samples were removed at intervals. Reactions were quenched by phenol extraction and DNAs were resolved by electrophoresis in a 20% gel cast and run in 40 mM Tris–acetate (pH 7.6), 1 mM EDTA, 20 mM KCl and 0.5 mM MgCl2. Lane a contains unrepaired G1 DNA; lanes b–l show reaction products sampled at 30, 60, 300, 600, 1200, 1800, 2700, 3600, 5400, 7200 and 10 800s. Lane m contains the 6mG-free 22wt quadruplex. (B) Time courses of representative repair reactions. Panels are labeled with the identities of the DNAs tested. Mole fractions of reactant and product bands were determined by fluorimetry. The smooth curves are fits of the time evolution of product formation, using Equation (4). Insets show the initial phases of each reaction in enlarged detail.
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Figure 7: Time course of alkyltransfer in G-quadruplex DNAs. (A) Reaction substrate and product detected by electrophoresis. AGT (4 μM) was mixed with G1 DNA (0.25 μM) to start the reaction and samples were removed at intervals. Reactions were quenched by phenol extraction and DNAs were resolved by electrophoresis in a 20% gel cast and run in 40 mM Tris–acetate (pH 7.6), 1 mM EDTA, 20 mM KCl and 0.5 mM MgCl2. Lane a contains unrepaired G1 DNA; lanes b–l show reaction products sampled at 30, 60, 300, 600, 1200, 1800, 2700, 3600, 5400, 7200 and 10 800s. Lane m contains the 6mG-free 22wt quadruplex. (B) Time courses of representative repair reactions. Panels are labeled with the identities of the DNAs tested. Mole fractions of reactant and product bands were determined by fluorimetry. The smooth curves are fits of the time evolution of product formation, using Equation (4). Insets show the initial phases of each reaction in enlarged detail.

Mentions: We took advantage of the observation (24) that short quadruplex DNAs containing 6mG residues migrate more slowly in native gel electrophoresis than do homologous quadruplexes lacking 6mG. Shown in Figure 7A, wild-type AGT added to solutions containing 6mG-quadruplex DNA gradually converts the low mobility (methylated) form into one with a higher mobility indistinguishable from that of the non-methylated 22wt DNA. In contrast, alkyltransfer-inactive C145A AGT does not produce a detectable shift (not shown). We interpret the mobility change as evidence of DNA repair. Mole fractions of repaired and unrepaired DNAs were quantified by fluorimetry; representative reaction time courses are shown in Figure 7B. The smooth curves are fits using Equation (4), which embodies a two-phase kinetic model:(4)\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{upgreek}\usepackage{mathrsfs}\setlength{\oddsidemargin}{-69pt}\begin{document}}{}\begin{equation*} {{F}} = {A}_1 (1 - \exp ( - {\rm k}_1 {t})) + {A}_2 (1 - \exp ( - {\rm k}_2 {t}))\end{equation*}\end{document}


Repair of O6-methylguanine adducts in human telomeric G-quadruplex DNA by O6-alkylguanine-DNA alkyltransferase.

Hellman LM, Spear TJ, Koontz CJ, Melikishvili M, Fried MG - Nucleic Acids Res. (2014)

Time course of alkyltransfer in G-quadruplex DNAs. (A) Reaction substrate and product detected by electrophoresis. AGT (4 μM) was mixed with G1 DNA (0.25 μM) to start the reaction and samples were removed at intervals. Reactions were quenched by phenol extraction and DNAs were resolved by electrophoresis in a 20% gel cast and run in 40 mM Tris–acetate (pH 7.6), 1 mM EDTA, 20 mM KCl and 0.5 mM MgCl2. Lane a contains unrepaired G1 DNA; lanes b–l show reaction products sampled at 30, 60, 300, 600, 1200, 1800, 2700, 3600, 5400, 7200 and 10 800s. Lane m contains the 6mG-free 22wt quadruplex. (B) Time courses of representative repair reactions. Panels are labeled with the identities of the DNAs tested. Mole fractions of reactant and product bands were determined by fluorimetry. The smooth curves are fits of the time evolution of product formation, using Equation (4). Insets show the initial phases of each reaction in enlarged detail.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150771&req=5

Figure 7: Time course of alkyltransfer in G-quadruplex DNAs. (A) Reaction substrate and product detected by electrophoresis. AGT (4 μM) was mixed with G1 DNA (0.25 μM) to start the reaction and samples were removed at intervals. Reactions were quenched by phenol extraction and DNAs were resolved by electrophoresis in a 20% gel cast and run in 40 mM Tris–acetate (pH 7.6), 1 mM EDTA, 20 mM KCl and 0.5 mM MgCl2. Lane a contains unrepaired G1 DNA; lanes b–l show reaction products sampled at 30, 60, 300, 600, 1200, 1800, 2700, 3600, 5400, 7200 and 10 800s. Lane m contains the 6mG-free 22wt quadruplex. (B) Time courses of representative repair reactions. Panels are labeled with the identities of the DNAs tested. Mole fractions of reactant and product bands were determined by fluorimetry. The smooth curves are fits of the time evolution of product formation, using Equation (4). Insets show the initial phases of each reaction in enlarged detail.
Mentions: We took advantage of the observation (24) that short quadruplex DNAs containing 6mG residues migrate more slowly in native gel electrophoresis than do homologous quadruplexes lacking 6mG. Shown in Figure 7A, wild-type AGT added to solutions containing 6mG-quadruplex DNA gradually converts the low mobility (methylated) form into one with a higher mobility indistinguishable from that of the non-methylated 22wt DNA. In contrast, alkyltransfer-inactive C145A AGT does not produce a detectable shift (not shown). We interpret the mobility change as evidence of DNA repair. Mole fractions of repaired and unrepaired DNAs were quantified by fluorimetry; representative reaction time courses are shown in Figure 7B. The smooth curves are fits using Equation (4), which embodies a two-phase kinetic model:(4)\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{upgreek}\usepackage{mathrsfs}\setlength{\oddsidemargin}{-69pt}\begin{document}}{}\begin{equation*} {{F}} = {A}_1 (1 - \exp ( - {\rm k}_1 {t})) + {A}_2 (1 - \exp ( - {\rm k}_2 {t}))\end{equation*}\end{document}

Bottom Line: Its functions with short single-stranded and duplex substrates have been characterized, but its ability to act on other DNA structures remains poorly understood.Here, we examine the functions of this enzyme on O(6)-methylguanine (6mG) adducts in the four-stranded structure of the human telomeric G-quadruplex.This distinction may reflect differences in the conformational dynamics of 6mG residues in G-quadruplex DNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, Lexington, KY 40536, USA.

Show MeSH
Related in: MedlinePlus