A Fix on AlkB
Authors: Davenport R. John 
Published: August 2003

After almost 2 decades of scrutiny, a mysterious DNA-repair protein has bared its inner workings. By mixing a set of ingredients never before known to be used by enzymes with that function, two research groups have deduced its mechanism. The studies reveal that the protein works differently from other DNA fix-it proteins and repairs defects in a single step. The widely conserved enzyme might fight damage that promotes aging.

The protein, AlkB from
Escherichia coli
, helps cells cope with DNA that's scarred by methyl groups, which can mutate genes or kill cells if they lie at particular sites. Researchers identified the gene that produces AlkB in 1983, and subsequent studies showed that cells lacking AlkB succumb readily to methyl-adding compounds. A clue about how the protein mends methylated DNA came last year, when computer models compared its sequence to the sequences of proteins with known function. According to the analysis, AlkB resembles a subset of dioxygenase enzymes that require iron and α-ketoglutarate, a small molecule that participates in metabolism, to do their jobs; no previously studied DNA-repair enzyme used these molecules. This group of dioxygenases adds oxygen to methyl groups; the combination then spontaneously falls off the substrate as formaldehyde.

Two teams, Falnes and colleagues and Trewick and colleagues, now independently show that AlkB performs in the manner that the models predicted. The researchers purified AlkB from
E. coli
cells and tested whether it freed radioactive methyl groups from DNA molecules that had been treated with methylating compounds. It did so only when reaction mixtures contained iron and α-ketoglutarate. The teams also showed that the reaction requires oxygen and generates formaldehyde. Some methyl-removing enzymes chop out the entire damaged base, relying on other proteins to fill in an unadulterated nucleotide. Other enzymes remove only the methyl group but do so by permanently attaching it to themselves, which prevents them from performing further reactions. AlkB is "a new example of direct reversal," says DNA-repair biologist Leona Samson of the Massachusetts Institute of Technology in Cambridge. It acts as a true enzyme, recycling to remove other methyl groups.

Besides establishing a mechanism, the scientists uncovered AlkB's natural targets and physiological role. AlkB attacked methyl groups that the methylating agents add to single-stranded, but not double-stranded, DNA. Both teams propose that AlkB helps cells recover from damage inflicted during DNA replication or transcription, when the two strands composing the double helix separate. Additional studies by Falnes and colleagues support that idea: Copying machinery normally stalls on methylated single-stranded DNA, but it didn't when the team first added the AlkB reaction mixture to the DNA.
"I think it's wonderful," says Samson. "It's a completely novel mechanism that's clean and efficient." Other organisms, including humans, carry similar proteins; the new studies will help scientists discern their function. Because oxidative stress encourages DNA methylation, Samson notes, AlkB might play an important role in aging; understanding the mechanism will aid researchers in deciphering exactly how. In addition, doctors commonly use methylating compounds in chemotherapy, and blocking AlkB might make cancer cells more vulnerable to such treatment. Having exposed some of AlkB's secrets, scientists can now devise ways to manipulate the protein.
--R. John Davenport

P. Ø. Falnes, R. F. Johansen, E. Seeberg, AlkB-mediated oxidative demethylation reverses DNA damage in
Escherichia coli
.
Nature
419
, 178-182 (2002).
[Abstract]
[Full Text]

S. C. Trewick, T. F. Henshaw, R. P. Hausinger, T. Lindahl, B. Sedgwick, Oxidative demethylation by
Escherichia coli
AlkB directly reverts DNA base damage.
Nature
419
, 174-178 (2002).
[Abstract]
[Full Text]