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Kinetic studies of Escherichia coli AlkB using a new fluorescence-based assay for DNA demethylation.

Roy TW, Bhagwat AS - Nucleic Acids Res. (2007)

Bottom Line: It may also be used to study other demethylation reactions including demethylation of histones.We used it to determine the kinetic constants for AlkB and found them to be somewhat different than previously reported values.The results show that AlkB demethylates 1mA and 3mC with comparable efficiencies and has only a modest preference for a single-stranded DNA substrate over its double-stranded DNA counterpart.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Wayne State University, Detroit, MI 48202, USA.

ABSTRACT
The Escherichia coli AlkB protein catalyzes the direct reversal of alkylation damage to DNA; primarily 1-methyladenine (1mA) and 3-methylcytosine (3mC) lesions created by endogenous or environmental alkylating agents. AlkB is a member of the non-heme iron (II) alpha-ketoglutarate-dependent dioxygenase superfamily, which removes the alkyl group through oxidation eliminating a methyl group as formaldehyde. We have developed a fluorescence-based assay for the dealkylation activity of this family of enzymes. It uses formaldehyde dehydrogenase to convert formaldehyde to formic acid and monitors the creation of an NADH analog using fluorescence. This assay is a great improvement over the existing assays for DNA demethylation in that it is continuous, rapid and does not require radioactively labeled material. It may also be used to study other demethylation reactions including demethylation of histones. We used it to determine the kinetic constants for AlkB and found them to be somewhat different than previously reported values. The results show that AlkB demethylates 1mA and 3mC with comparable efficiencies and has only a modest preference for a single-stranded DNA substrate over its double-stranded DNA counterpart.

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Related in: MedlinePlus

Absorbance and fluorescence properties of APADH. (A) Fluorescence intensity (arbitrary units) as a function of concentration. The ordinate is from 0 to 1000 U because this is the maximum range of the spectrophotometer. The excitation at 363 nm and emission was at 482 nm. Fluorescence intensity was determined at two different photomultiplier voltages—800 V (filled triangle); 700 V (filled square). Mean intensity and standard deviation from three independent measurements is shown. (B) APADH absorbance at 363 nm as a function of concentration. The ordinate is from 0 to 1.0 because this is the range in which absorbance can be meaningfully measured using most spectrophotometers.
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Figure 2: Absorbance and fluorescence properties of APADH. (A) Fluorescence intensity (arbitrary units) as a function of concentration. The ordinate is from 0 to 1000 U because this is the maximum range of the spectrophotometer. The excitation at 363 nm and emission was at 482 nm. Fluorescence intensity was determined at two different photomultiplier voltages—800 V (filled triangle); 700 V (filled square). Mean intensity and standard deviation from three independent measurements is shown. (B) APADH absorbance at 363 nm as a function of concentration. The ordinate is from 0 to 1.0 because this is the range in which absorbance can be meaningfully measured using most spectrophotometers.

Mentions: To detect formaldehyde released during the demethylation of DNA, we converted it to formate using FDH. During this conversion NAD+ is converted to NADH, and the latter molecule is usually detected based on its UV absorbance (17). In our experiments, we used APAD+ instead of NAD+ as the cofactor for FDH because of its higher redox potential leading to greater stability of the reduced form at various pHs and a higher extinction coefficient which contributes to greater sensitivity (18). We also chose to use fluorescence properties APADH to develop the demethylation assay because of its greater sensitivity over UV absorbance by a factor of ∼30 (Figure 2). Consequently, we were able to lower the amount of methylated DNA base used for the assay from several nanomoles—for a typical UV-absorbance assay—to subnanomole amounts. This allowed us to use synthetic oligonucleotides containing methylated bases in a defined sequence context as substrates for kinetic studies. Most previous studies have used randomly methylated poly-dA (or poly-dC) or DNAs containing both 1mA and 3mC for their assays (2,9,13,14,19,20).Figure 2.


Kinetic studies of Escherichia coli AlkB using a new fluorescence-based assay for DNA demethylation.

Roy TW, Bhagwat AS - Nucleic Acids Res. (2007)

Absorbance and fluorescence properties of APADH. (A) Fluorescence intensity (arbitrary units) as a function of concentration. The ordinate is from 0 to 1000 U because this is the maximum range of the spectrophotometer. The excitation at 363 nm and emission was at 482 nm. Fluorescence intensity was determined at two different photomultiplier voltages—800 V (filled triangle); 700 V (filled square). Mean intensity and standard deviation from three independent measurements is shown. (B) APADH absorbance at 363 nm as a function of concentration. The ordinate is from 0 to 1.0 because this is the range in which absorbance can be meaningfully measured using most spectrophotometers.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Absorbance and fluorescence properties of APADH. (A) Fluorescence intensity (arbitrary units) as a function of concentration. The ordinate is from 0 to 1000 U because this is the maximum range of the spectrophotometer. The excitation at 363 nm and emission was at 482 nm. Fluorescence intensity was determined at two different photomultiplier voltages—800 V (filled triangle); 700 V (filled square). Mean intensity and standard deviation from three independent measurements is shown. (B) APADH absorbance at 363 nm as a function of concentration. The ordinate is from 0 to 1.0 because this is the range in which absorbance can be meaningfully measured using most spectrophotometers.
Mentions: To detect formaldehyde released during the demethylation of DNA, we converted it to formate using FDH. During this conversion NAD+ is converted to NADH, and the latter molecule is usually detected based on its UV absorbance (17). In our experiments, we used APAD+ instead of NAD+ as the cofactor for FDH because of its higher redox potential leading to greater stability of the reduced form at various pHs and a higher extinction coefficient which contributes to greater sensitivity (18). We also chose to use fluorescence properties APADH to develop the demethylation assay because of its greater sensitivity over UV absorbance by a factor of ∼30 (Figure 2). Consequently, we were able to lower the amount of methylated DNA base used for the assay from several nanomoles—for a typical UV-absorbance assay—to subnanomole amounts. This allowed us to use synthetic oligonucleotides containing methylated bases in a defined sequence context as substrates for kinetic studies. Most previous studies have used randomly methylated poly-dA (or poly-dC) or DNAs containing both 1mA and 3mC for their assays (2,9,13,14,19,20).Figure 2.

Bottom Line: It may also be used to study other demethylation reactions including demethylation of histones.We used it to determine the kinetic constants for AlkB and found them to be somewhat different than previously reported values.The results show that AlkB demethylates 1mA and 3mC with comparable efficiencies and has only a modest preference for a single-stranded DNA substrate over its double-stranded DNA counterpart.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Wayne State University, Detroit, MI 48202, USA.

ABSTRACT
The Escherichia coli AlkB protein catalyzes the direct reversal of alkylation damage to DNA; primarily 1-methyladenine (1mA) and 3-methylcytosine (3mC) lesions created by endogenous or environmental alkylating agents. AlkB is a member of the non-heme iron (II) alpha-ketoglutarate-dependent dioxygenase superfamily, which removes the alkyl group through oxidation eliminating a methyl group as formaldehyde. We have developed a fluorescence-based assay for the dealkylation activity of this family of enzymes. It uses formaldehyde dehydrogenase to convert formaldehyde to formic acid and monitors the creation of an NADH analog using fluorescence. This assay is a great improvement over the existing assays for DNA demethylation in that it is continuous, rapid and does not require radioactively labeled material. It may also be used to study other demethylation reactions including demethylation of histones. We used it to determine the kinetic constants for AlkB and found them to be somewhat different than previously reported values. The results show that AlkB demethylates 1mA and 3mC with comparable efficiencies and has only a modest preference for a single-stranded DNA substrate over its double-stranded DNA counterpart.

Show MeSH
Related in: MedlinePlus