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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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Kinetics of AlkB for DNA with 1mA. (A) Initial reaction velocities of AlkB (V) with SS DNA oliogonucleotide containing a single 1mA as a function of substrate concentration [S]. Mean and standard deviation from three or more separate identical reactions are shown. (B) Similar data with DS substrate containing a single 1mA. Inset—the data re-plotted with [S]/V as a function of [S].
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Figure 4: Kinetics of AlkB for DNA with 1mA. (A) Initial reaction velocities of AlkB (V) with SS DNA oliogonucleotide containing a single 1mA as a function of substrate concentration [S]. Mean and standard deviation from three or more separate identical reactions are shown. (B) Similar data with DS substrate containing a single 1mA. Inset—the data re-plotted with [S]/V as a function of [S].

Mentions: Four substrates were used for studying AlkB kinetics. Two of them contained a 29-base DNA oligomer with a single 1mA and the other two substrates had the same base sequence but contained instead one 3mC. Unmethylated complementary DNAs were hybridized to the methylated DNAs to create the DS substrates and both SS and DS versions of the methylated DNAs were used for the demethylation assays. The initial velocities of APADH production from three or more independent reactions for five or six concentrations of substrates are shown in Figure 4 and Supplementary Figure S3.Figure 4.


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

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

Kinetics of AlkB for DNA with 1mA. (A) Initial reaction velocities of AlkB (V) with SS DNA oliogonucleotide containing a single 1mA as a function of substrate concentration [S]. Mean and standard deviation from three or more separate identical reactions are shown. (B) Similar data with DS substrate containing a single 1mA. Inset—the data re-plotted with [S]/V as a function of [S].
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Kinetics of AlkB for DNA with 1mA. (A) Initial reaction velocities of AlkB (V) with SS DNA oliogonucleotide containing a single 1mA as a function of substrate concentration [S]. Mean and standard deviation from three or more separate identical reactions are shown. (B) Similar data with DS substrate containing a single 1mA. Inset—the data re-plotted with [S]/V as a function of [S].
Mentions: Four substrates were used for studying AlkB kinetics. Two of them contained a 29-base DNA oligomer with a single 1mA and the other two substrates had the same base sequence but contained instead one 3mC. Unmethylated complementary DNAs were hybridized to the methylated DNAs to create the DS substrates and both SS and DS versions of the methylated DNAs were used for the demethylation assays. The initial velocities of APADH production from three or more independent reactions for five or six concentrations of substrates are shown in Figure 4 and Supplementary Figure S3.Figure 4.

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