Monster Made Good
Of obvious importance to cells everywhere
is the maintenance of the library of instructions for doing... everything(!).This
library is made entirely of DNA. Indeed, the complexity of the human body
and nervous system could NOT BE ACHIEVED if the error rate of DNA replication
were even 10-fold higher. Amongst the key players in error prevention
and correction are the machines that do the actual copying of DNA, the
Despite their critical roles, if we look carefully at these machines,
we can see that they can be viewed as cobbled together pieces of other
and crippled derivatives of the RNA Polymerases from which they may have
Big Job:Adding nucleotides
How does Polymerase know which nucleotide
to add? The short answer is, it doesn't. There is no reason to believe
that DNA Polymerase ever achieves direct knowledge of the nucleotide it
Indeed, it has been elegantly demonstrated that it can add a slew of nucleotides
outside of the canonical Big 4 it's used to. How, then, does it achieve "chemically
rates of error?
The first secret lies
in the template--the
existing strand of DNA being used to generate a complementary copy.
By incorporating the
DNA into its active site, DNA Polymerase uses the template nucleotide
to assess the appropriateness of a given dNTP for addition.In
short, DNA Polymerase never knows what it's adding, but it does determine
whether its candidate 'fits' --as defined by the ability of the newcomer
to form a specifically predicted structure with it. Recall that G:::C
and A::T basepairs are
superimposable, despite the different shapes and hydrogen bonding capabilities
of their constituents. Thus DNA Polymerase may beignorant of the basepairing
'code' wherein G calls C, A calls T and vice versa, but itdetects
whether 'whatever the template nucleotide is' matches 'whatever the
incoming nucleotide is'. One direct prediction of this model is that DNA
Polymerase can add ANY purine and any pyrimidine as long as the two
bonds!This prediction is indeed met [Figure
of synthetic ntes that Pol will add)...].Indeed,
if Martian DNA were to be found, our DNA Polymerase should be able
and sequence it whether or not it uses the same 4 bases that we do--as
long as the backbone is deoxyribose and as long as nothing too bulky
is hanging off the base rings.
nucleotides: Polymerase is lazier than you think!
One common misconception is that DNA Polymerase
'chooses' or 'selects' the desired nucleotide from solution. Quite the
contrary, ANYthing can wander into the active site of DNA Pol! To grab
a specific nucleotide,
DNA Pol would have to 1) form an opinion about what it wanted, and 2)
have a machinery that lassoed nucleotides (this machinery would need to
take instruction as to what DNA Pol wanted AND could see or smell nearby
nucleotides!). To the contrary, the open binding site of DNA Polymerase
is like an available
Pol with just a funnel as a binding site, with basepairing positions
visible at bottom]. There
will, however, be constraints on what nucleotides stay in the
pocket. DNA polymerase must check to make sure it is not polymerizing
ribonucleotides. How can it 'check'? By creating a binding pocket that
with the structure of a ribonucleotide. You can imagine designing the
pocket in DNA Pol such that a deoxyribonucleotide fit snugly once basepaired,
but that the extra hydroxyl at the 2' position of a ribonucleotide bumped
into some part of the protein, preventing it from 'assuming the position.'
Conversely, expected features of correct molecules could be 'rewarded'
by designing the DNA Pol pocket such that favorable interactions occurred
with, for example, the ribose, the triphosphate, the 3' OH--IF they had
been oriented by favorable hydrogen bonding of the base on the incoming
nucleotide to the template [again,
having a nucleotide or couple shown in several orientations with the
one showing interactions of its phosphates, ribose, etc. to sites on Pol].
So all small molecules check in--but only basepairable deoxynucleotide
stay the night and are added to the growing chain.
Assessing the fit
How can DNA Pol come to know that a nucleotide is in
the right position? Consider how an enzyme works. It acts by aligning
the key players in whatever reaction it is catalyzing. By taking a somewhat
'hands off' approach, DNA Pol can make the interactions of the candidate
nucleotide much more important. The newcomer becomes DEPENDENT upon its
own interactions (H-bonding, stacking with other nucleotides), and if
these interactions are less than optimal, the staying power of the guest
nucleotide suffers. DNA Pol can be built such that its catalytic assistance
only comes to bear if the nucleotide assists by presenting its triphosphates
for optimal attack by the 3'OH on the primer strand.
Since the materials it works with are
constantly undergoing chemically driven changes that alter the hydrogen
surface they present (see section on tautomerization),
DNA Pol's strategy of adding bases by fit and feel is doomed fail from
time to time. Errors WILL occur. How is it we nonetheless exist with
our 30,000-40,000 genes? The answer lies in the parts and properties of
order to deal with the inevitable sidetrips of nucleotides to their
forms, two 'readings' of the basepairing ability of a nucleotide are made.
First is the requirement for 'fit' before a candidate nucleotide is
added as discussed above.
However, since their is a possibility that either one is 'lying' about
its identity--by adopting the uncommon tautomeric form--this step alone
to achieve high fidelity. By 'checking it twice', DNA Pol takes advantage
of the fact that the tautomeric forms are energetically less favorable
than their more common counterparts, and thus dependably short-lived.
After making an addition to the chain, DNA Polymerase is now in a
to use the new base as the site of the subsequent addition. However, it
only makes an addition if the previously added base is well-behaved,
i.e. firmly basepaired.If the previous
nucleotide is not basepaired, it will occupy a variety of positions,
and only rarely
simulate a basepaired nucleotide. This pickiness is the first step in
error correction by DNA Polymerase.
Sounds good in words. But how to do this mechanically?
By making DNA Pol somewhat 'helpless' in terms of positioning the primer
nucleotide. Again, weakness is accuracy! If DNA Pol is not too 'pushy'
in terms of forcing the primer nucleotide (most critically: the 3' OH
of the ribose!), the nucleotide must hold itself in place. It's going
to need all the help it can get--including the basepairing H-bonds! Failure
to get everything lined up will mean DNA Pol cannot proceed--the key
chemical groups just won't be in the right places for catalysis.
what profit does a DNA Pol gain by 'stalling' after making an incorrect
If it were working on its own, none--eventually the machine would either
fall off the DNA, ending the cell's hopes of replicating its genome,
or the incorrect
addition might through chance 'sit still' in the appropriate position
long enough to allow subsequent additions. But somewhere in the course
history, a happy shotgun marriage was performed: the
joining of a DNA Polymerase with a non-specific exonuclease (a machine
lops off nucleotides from the end of a chain). [attaching
Pac-Man nuclease by a stiff spring to nuclease could capture the lack
While this seems a silly idea for an addition to a DNA Polymerase molecule,
in the context of proofreading it makes brilliant sense. By guaranteeing
the presence of nucleotide removal machinery in the vicinity of DNA polymerase,
the cell gains the ability to resolve the dilemma presented above. After
stalling of polymerase due to an incorrect addition, opportunities arise
for the exonuclease
to 'get its hands on' the aberrant nucleotide. The nuclease can be thought
of as a ravening beast--it eats what it finds--but due to the propensity
of DNA Pol to stall after an erroneous addition, a reasonably slick scenario
for error correction emerges:
Polymerase is fooled by a tautomeric form or by chance apparent 'fit' of an
incorrect nte. in the correct position.
proceeding to make the next addition, DNA Pol is unable to 'find' a 3'
in the correct position for further polymerization. Further additions
are thus blocked
Pol slides back and forth on the 'rails' of DNA aimlessly
the DNA Pol will slide 'backwards', exposing the recently added nucleotides to
DNA 3' end will intermittently 'fray' (separate from the template strand),
and the freed strand of DNA (capped by the
incorrect nucleotide!) will wander
wandering strand may wander into the maw of the nuclease, which snips off
as many ntes. as it has the opportunity to
lacking the offending mis-addition, there will come a time when the DNA
're-zip' (again, driven only by chance!) and DNA Pol wanders into position
and is happily confronted with a correct pairing, to which it merrily
adds and the
polymerization process continues.
of the above would be astoundingly excellent; however it'd be pretty complex.
Perhaps mini-cartoons of each step? Can have the ole 'thought bubble' coming
out of DNA Pol containing a question mark when it can't proceed, an exclamation
point in 7. A ticking watch somewhere for pauses could illustrate the VERY
KEY point that passage of time drives this]
The 'discovery' of proofreading by DNA
polymerases illuminates several important principles and practices of evolution
of protein machines. First, note that the story as told contains an inherent
paradox--if DNA stalling came first, then there would have been no DNA synthesis
until accidental addition of the exonuclease to the complex--doubtless the
organism would be extinct long before the opportunity arose!
A more likely scenario is that original
DNA Polymerases were not so picky about placement of the 3'OH. How could this
be? Firstly, note that RNA Polymerases are not
so picky about template-directed positioning of the 3'OH. Doubtless they contain
structures that draw or hold the 3' nucleotide in the correct position for
addition and thus do not rely on basepairing to perform this task. Ancient
DNA Pols evolved from RNA Pols would be expected to share this property. Accidental
addition of a nuclease to the complex at this point would still have benefited
DNA Pol. Though stalling may not have been absolute, an incorrect addition
will inevitably be 'harder' to position than a correct one. So some stalling
is still expected. By creating the capability to resolve the situation as
described above, a more accurate and efficient DNA Pol would be created.
comes the cool part. To make DNA Pol more accurate once the nuclease has been
'glued on', we actually need to cripple the DNA Polymerase! By decayingits
ability to 'grasp' the last-added nte., we render the stalling at mis- additions
ever more lengthy, and the events leading up to nuclease action more inevitable.
Eventually we would achieve the current day situation in which many DNA Polymerases
are extremely picky about behavior of last-added nucleotides and thus extremely
accuratesince the inevitable outcome
is removal of the offending nucleotide.
model also explains another property of DNA Polymerases--their inability
initiate nucleotide chains using template alone. Is this pickiness inevitable
in a nucleotide polymerase? Clearly not--RNA Polymerases are quite capable
of doing the "1+1" reaction, adding the first two nucleotides together
to initiate a new nucleotide strand. Why then would DNA Pols, which presumably
evolved FROM RNA Pols, lack this capability? First, it may be an inevitable
outcome of primer dependance--by loosening DNA Pol's 'grip' on the template
nucleotide, you would expect to lose the ability to hold a single nucleotide
in the same position (such as would be required to perform the "1+1" initial
addition to start the chain).
second argument for why DNA Pol would lose the ability to initiate synthesis
without a primer is this:such an ability
would defeat proofreading! Note how the sequence of events in proofreading
requires that DNA Pol stall after a mis-addition. This represents a window
of opportunity for the nuclease to get involved. If, on the other hand, DNA
Pol had the capacity to initiate a new chain, it would simply leave its mistake
behind and move on--doing a nice job of moving quickly, and a very poor one
of moving accurately.