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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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Tris is a substrate for FDH. Utilization of Tris as a substrate by FDH is detected by the coupled conversion of APAD+ to APADH. Fluorescence intensity of resulting APADH is shown as a function of time. No AlkB or DNA was present in these reactions. Reactions with no Tris (filled circle), 0.1 mM Tris (open triangle), 1 mM Tris (open circle) and 5 mM Tris (open square) are shown.
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Figure 3: Tris is a substrate for FDH. Utilization of Tris as a substrate by FDH is detected by the coupled conversion of APAD+ to APADH. Fluorescence intensity of resulting APADH is shown as a function of time. No AlkB or DNA was present in these reactions. Reactions with no Tris (filled circle), 0.1 mM Tris (open triangle), 1 mM Tris (open circle) and 5 mM Tris (open square) are shown.

Mentions: During some preliminary experiments, we discovered that the commonly used buffering agent, Tris, was a substrate for FDH and led to conversion of APAD+ to APADH in the absence of AlkB or methylated DNA substrate in the reaction (Figure 3). This is probably because FDH also acts on some alcohols (21). Tris contains three primary alcohol groups that may be converted to acidic form by FDH. To avoid the contribution of this reaction to background fluorescence, HEPES was used as buffering agent in the reactions instead of Tris.Figure 3.


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

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

Tris is a substrate for FDH. Utilization of Tris as a substrate by FDH is detected by the coupled conversion of APAD+ to APADH. Fluorescence intensity of resulting APADH is shown as a function of time. No AlkB or DNA was present in these reactions. Reactions with no Tris (filled circle), 0.1 mM Tris (open triangle), 1 mM Tris (open circle) and 5 mM Tris (open square) are shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175350&req=5

Figure 3: Tris is a substrate for FDH. Utilization of Tris as a substrate by FDH is detected by the coupled conversion of APAD+ to APADH. Fluorescence intensity of resulting APADH is shown as a function of time. No AlkB or DNA was present in these reactions. Reactions with no Tris (filled circle), 0.1 mM Tris (open triangle), 1 mM Tris (open circle) and 5 mM Tris (open square) are shown.
Mentions: During some preliminary experiments, we discovered that the commonly used buffering agent, Tris, was a substrate for FDH and led to conversion of APAD+ to APADH in the absence of AlkB or methylated DNA substrate in the reaction (Figure 3). This is probably because FDH also acts on some alcohols (21). Tris contains three primary alcohol groups that may be converted to acidic form by FDH. To avoid the contribution of this reaction to background fluorescence, HEPES was used as buffering agent in the reactions instead of Tris.Figure 3.

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