Paper Up: Correlations between DNA binding thermodynamics and DNA polymerase activity

Our lab has a paper called: "Enthalpic Switch-Points and Temperature Dependencies of DNA binding and Nucleotide Incorporation by Pol I DNA Polymerases" that was just published in BBA (Biochimica et Biophysica Acta): Proteins and Proteomics. The study follows up on an observation and prediction we had made some years ago in a different paper.

The study deals with quite a lot of rather detailed thermodynamics of DNA binding (free energy, enthalpy, entropy, heat capacity…) and looks for correlations between such thermodynamic measurements of binding and the functional behavior of a couple of similar DNA polymerases. DNA polymerases are the enzymes that replicate DNA. The strong correlation that we found was between entropy (ΔH) of binding and onset of functional replication activity. Or to state it another way:

As the temperature increases, the enthalpy of binding of the polymerases to DNA goes from positive (unfavorable = heat input required) to negative (favorable = heat released). Right at the temperature where the binding enthalpy switches from positive to negative, the replication activity of the polymerase effectively switches "on". So, the polymerases bind to DNA quite fine at lower temperatures, but are effectively "shut off" functionally until after the temperature where the ΔH of binding switches from positive to negative. And this temperature is not the same for each polymerase – it is specific to the particular polymerase. This correlation is illustrated in the figure below:


So what does this mean?  We believe it means that the simple binding of an enzyme to DNA is not sufficient for that enzyme to carry out its catalytic function on the DNA, and that the balance of different types of binding energy (enthalpy versus entropy) may dictate when a bound enzyme is turned "off" versus turned "on".  While this is the only set of DNA binding proteins where applicable overlapping binding and activity data have been compared, we hypothesize that this correlation may hold for many DNA-binding enzymes.

It is also interesting because a few other studies in some very different systems have also recently been finding that for some processes, following the enthalpy is much more important than following the free energy (ΔG) (the free energy is what biochemists "follow" for mechanistic clues 99% of the time).  We mention other examples in the paper, but a particularly interesting one is the finding that the efficacy of HIV protease inhibitors also tracks with enthalpy of binding, and not with the free energy of binding (Freire, Drug Discovery Today 13:19 (2008)).

So why does switching from positive to negative enthalpy effectively switch "on" the protein activity?  Good question, we'd love to know the answer.  We postulate a few possibilities in the paper – one of which being that maybe the catalytic reactions just need a little heat, and a negative binding enthalpy will release a little heat.

The data in our paper also speak to a longstanding concept in extremophile research: something called the concept of "corresponding states".  This concept states that a protein from a thermophile (a high temperature organism) will function "normally" only at high temperatures.  According to the corresponding states theory, our thermophilic polymerase should not bind DNA at all at lower temperatures, and should not show "normal" nucleotide activity until quite high temperatures.  Our data show that neither of these "corresponding states" predictions is true, and suggest that the situation is somewhat more complicated than "heat up the protein and then it will behave like the lower temperature version of the protein".  The corresponding states theory has been challenged by a few different studies over the past decade, but it turns out that for a variety of reasons it is actually difficult to get appropriate data to properly refine it.  It is one of those theories that must wait a little longer for experimental technology to catch up to it for it to be appropriately tested and refined.

As always, if would like a copy of the paper and you cannot get it free from the link above or your library, I will send you one.

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Dear Prof. LiCata,
Thanks a lot for this popular explanation of your article. It became a bit clearer now.
However, when I read your article I was looking for the answer whether overall stability of primer-template duplex may determine the likelihood that the polymerase binds to a duplex.
Do I understand correctly, that you studied only perfectly matched DNA duplex?