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Combining chemistry with biotechnology, Saulius
Klimasauskas, a Howard Hughes Medical Institute (HHMI) international
research scholar at the Institute of Biotechnology in Vilnius,
Lithuania, and chemists at the Institute of Organic Chemistry in
Aachen, Germany, have harnessed a group of essential enzymes to add
various chemical groups to DNA, thereby altering its function. The
work was published in an early online publication on November 27, 2005
in Nature Chemical Biology.
The enzymes at the heart of the study, known as DNA
methyltransferases, are one of the tools cells use to turn genes on
and off. By adding a simple cluster of four atoms -- a carbon atom
attached to three hydrogens, known to chemists as a methyl group -- to
specific bases within a DNA sequence, methyltransferases can
effectively shut a gene off. Methylation plays an important role in
embryonic development, genomic imprinting, and carcinogenesis because
it regulates gene expression.
Methyltransferases require a source for the methyl
groups that they attach to DNA, and most often that source is a
molecule called S-Adenosyl-L-methionine (AdoMet), sometimes known as
SAM or SAMe. Methyltransferases grab the methyl group from AdoMet and
transfer it directly to DNA, positioning it with enviable specificity
within the sequence. This specificity suggests that the enzymes can be
a useful tool in the laboratory. But Klimasauskas and colleagues
wanted the flexibility to attach more than just a simple methyl group.
In this study, the scientists demonstrated that
methyltransferases can indeed be used to transfer larger chemical
groups to large DNA molecules, in the same sequence-specific manner.
To try out their technique, the scientists
synthesized molecules that mimicked AdoMet, but had chemical groups
with longer carbon chains in the position where the methyl group was
usually located. The enzymes were able to grab the bulkier group and
transfer it to DNA. Since the family of DNA methyltransferases
includes enzymes capable of recognizing over 200 distinct sequences,
this new approach provided an unprecedented ability to manipulate DNA
experimentally.
To demonstrate the technique's potential to alter
DNA function, the researchers modified DNA in a position that blocked
another enzyme's ability to snip the molecule at its target site. "No
one has really thought about possible applications [of this] before
because no one thought it was possible,' said Klimasauskas. He
predicts that DNA methyltranferases will become a standard laboratory
tool like restriction endonucleases.
Earlier studies had suggested that the transfer of
chemical groups larger than a methyl group would not be possible, in
part because replacing AdoMet's methyl group lowered the chemical
reactivity of the compound. To overcome this problem, the authors took
some tips from organic chemistry textbooks and stabilized the transfer
with a multiple carbon bond.
"It turned out that our first bet, a double or
triple carbon-carbon bond, placed next to the transferable carbon unit,
helped to alleviate the problems that had plagued the reaction in
previous studies," Klimasauskas said. He likened the chemical reaction
to a mechanical spring, explaining that the chemical energy trapped in
AdoMet is sufficient to deliver a small methyl group to its target
compound. But delivering a larger compound required an auxiliary
"spring" to ensure it would reach the target. So, he said, "chemical
thinking" helped resolve the problematic enzymatic reaction.
"By demonstrating the transfer of carbon chains as
long as 4 to 5 units, we provide proof of principle that further
extensions should also be tolerated," Klimasauskas said.
Due to their sequence-specific nature, the
scientists found that methyltransferases have a distinct advantage
over other commonly used labeling techniques for DNA and other
biopolymers. "Our approach allows labeling of large native DNA
molecules at specific internal or terminal loci," Klimasauskas
explained.
While potential applications are many, the
researchers next plan to synthesize new AdoMet analogs to expand the
collection of chemical groups that can be transferred to DNA by
methyltrasferases. Klimasauskas's group is currently working to append
useful functional groups to extended chains. For example, researchers
often label cellular components with a molecule called biotin, because
it binds tightly to another molecule, streptavidin, and thus
streptavadin can be used to retrieve the molecule of interest. If
biotin were built into an AdoMet analog, Klimasauskas said, it could
then be used as a molecular hook to fish out all molecules that would
naturally be methylated in the cell. "There is no comparable way for
global analysis of the methylation targets in the cell," Klimasauskas
observed.
DNA is not the only molecule that is naturally
methylated in the cell - RNA and proteins also undergo methylation,
and the enzymes that carry out these reactions also rely on AdoMet as
their methyl source. Since the chemistry is the same, this technique
is likely to be applicable to those biomolecules as well, further
expanding its utility. Klimasauskas said that one potential
application might be to label various sites in the ribosome - the
RNA-based site of protein production -- with bright fluorophores using
appropriate RNA methyltransferases, enabling real-time dynamic studies
of the complicated mechanism of protein translation. |
