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Expanding the Genetic Code
Another area of research in the group involves expanding
the genetic code by adding additional letters (bases) to the
genetic alphabet.
This concept is simple, but putting it into practice is
not. To add bases, a scientist must have polymerase enzymes
that can recognize the unusual bases and replicate them with
the four standard bases—something that polymerases were
not designed by nature to do.
Romesberg and his group have been tackling this challenge
by using a combination of mutagenesis and phage display to
invent polymerases that can recognize additional bases.
The lab has designed DNA polymerases using a innovative
phage system that involves placing variant polymerase enzymes
on a phage particle next to a piece of DNA with unnatural
bases attached. The polymerases that are able to replicate
the DNA will do so, incorporating biotin labels at the same
time. These labels can then be used to distinguish the functional
mutants from the rest of the polymerases, which cannot replicate
the DNA with the unnatural bases.
And it works.
As a proof of principle, Romesberg's group, in collaboration
with the group of Professor Peter Schultz, evolved a DNA polymerase
into an RNA polymerase, demonstrating that directed evolution
could be used to make an enzyme with a new function.
"Our evolved mutants synthesize RNA as efficiently as the
DNA polymerase synthesize DNA," says Romesberg.
The group is currently working toward evolving a DNA polymerase
that can highly efficiently replicate DNA with unnatural nucleosides.
The team has worked with a number of novel bases, including
many that form complementary pairs because of hydrophobic
forces (as opposed to the hydrogen bonds that keep normal
bases together). Lately, they have also been designing novel
perfluorinated hydrocarbon bases, which have the strange property
of preferring to form their own liquid phase, unlike most
other substances, which are either soluble in water or oil.
The goal of this research is to develop an in vivo
system—a strain of bacteria with the mutant polymerase
that is able to use these unnatural bases and replicate, for
instance.
So far, this can only be done in vitro, and Romesberg and
his colleagues are working out the conditions that will allow
it to work in vivo as well. This involves determining
whether the unnatural nucleosides can be taken up into cells
and be transported to the right place, as well as being processed
into nucleotides along the way by metabolic enzymes which
add phosphates.
The Mutation is the Message
The last area of research in Romesberg's laboratory is,
as he puts it, "an effort to understand the genes that drive
evolution."
This is an odd way of stating the problem, perhaps, because
people have always understood evolution to be the force that
drives genes. Mutations introduced into the DNA of an individual
would propagate through the generations and eventually become
part of the germline if the mutation was of benefit to the
organism.
But most mutations are not beneficial to an organism, and
life has generally evolved to make as few as possible. One
of the main controls is the ubiquitous polymerase molecule,
which has the task of replicating the genome.
Evolution has provided life with the ability to replicate
genomes extremely well, with multiple, redundant repair and
proofreading mechanisms that would make even NASA jealous.
Nevertheless all organisms are subject to a certain level
of spontaneous mutations—mistakes that escape repair
and become part of the DNA of the cell in which they occur.
Slowly over time, these mutations accumulate and species diverge.
However, mutations can be fast-tracked as well. When cells
are subjected to ultraviolet radiation, for instance, the
rate of mutations increases because the energy of the light
is enough to cause bases of DNA to cross-link and introduce
errors. When the DNA is later replicated, these errors are
propagated into mutations.
However, this may not be the only way that UV radiation
causes mutations. In an experimental result that Romesberg
calls "amazing" mutations may actually increase because of
the cells' response to the radiation and not solely from the
radiation itself.
The experiment is a simple one: researchers take healthy
phage particles—a virus that infects bacteria—and
infect bacterial cells that have been previously subjected
to UV radiation. As you would expect, the DNA of the bacteria
is mutated because of its exposure to the radiation. However,
the results of the experiment show that the DNA of the virus
is also mutated. Since the viral DNA was never subjected to
UV radiation, it had to have been mutated by the cell.
What the experiment may be pointing to is that cells may
have a way to mutate themselves when they need to—such
as when they are threatened with extinction.
Take the bacterium Escherichia coli for instance.
When E. coli cells are subjected to damage, they upregulate
repair enzymes, which then go to work trying to fix the problem.
If the damage persists, the cell upregulates recombination
enzymes, which are tasked with recombining the DNA—another
way to repair it.
And, says Romesberg, if the damage still persists, the cells
upregulate enzymes whose sole task is to make mutations. Presumably
this is an effective evolutionary strategy for dealing with
environmental changes that might otherwise wipe them out.
In order to evolve, organisms have to mutate, so they turn
on the mutation process when they are threatened with extinction.
"There is a growing belief now that all organisms have evolved
these mechanisms to facilitate evolution when they have to,"
says Romesberg. "We are now working to understand these processes
at the biochemical level."
Romesberg's group, in collaboration with Associate Professor
Elizabeth Winzeler, is using a complicated competitive growth
assay involving the yeast Saccharomyces cerevisiae,
to do this.
They use a yeast library where every gene was deleted one
by one and replaced with a tag. The assay screens the yeast
for genes that are not necessary for growth but whose deletions
makes the yeast resistant to mutations when irradiated. Then
they can search for human homologues of these genes. Using
a similar screen, they have recently identified six genes
involved in the DNA damage response.
"That's what I love about science," says Romesberg as he
describes the discovery. "Being on the steep edge of the learning
curve."
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