Re: Why can't we use DNA polymerase III for PCR?

Hi Anisha,

I'm sorry that it has taken us a bit longer than usual to answer your question, but I hope that this answer is satisfactory for you.

You asked why we use the Klenow fragment of the DNA Polymerase I (Pol I) enzyme for PCR instead of the DNA Polymerase III (Pol III) enzyme. This is a good question, because as you point out, Pol III is much more processive than Pol I and already lacks a 5' to 3' exonuclease activity. In fact, Pol III is the principle enzyme used in Bacterial cells for replicating chromosomal DNA, whereas Pol I is used primarily to excise the RNA primer and fill in the resulting single-stranded regions of DNA. As a result, Pol I does not have to be nearly as processive as Pol III. In addition, Pol III operates about 100-fold faster than Pol I, incorporating 1000 nucleotides per second to Pol I's 10 nucleotides per second.

However, the key difference between the utility of these enzymes for PCR lies in those very functions. Pol I only has to hold on to the DNA molecule for a short amount of time, and as a result, Pol I is a monomer. Pol III on the other hand, has to hold onto the DNA molecule through millions of nucleotide additions, and it has to replicate both stands of the molecule at the same time, where Pol I only has to replicate one strand at a time. As a result, the Pol III enzyme is a multi-subunit complex that forms an asymmetric dimer (and is therefore known as a holoenzyme). Whereas Pol I is a single protein molecule, Pol III is a complex of 22 individual peptides that fall into 10 distinct peptide cateogies.

Compare the structures of Pol I and Pol III, below.

DNA Polymerase I	DNA Polymerase III

So, the reason that Pol III is unsuitable for PCR is two-fold. First, it would be very difficult to reconstitute the activity of Pol III in vitro for use in PCR -- we would have to include 10 different subunits in the appropriate stoichiometries, and then have them all assemble into complete, active Pol III complexes every time someone wanted to do PCR. In addition, even if you could manage to assemble active Pol III complexes at the start of every PCR, it seems to me that it would be difficult to maintain those functional complexes through the several dozen rounds of extreme temperature fluctuation that take place in a typical PCR.

Second, Pol III catalyzes the replication of both strands of the DNA molecule at the same time, starting from the same replication point. However, in PCR, you want to start replication on each strand from different replication points. In this respect, PCR is much more similar to the normal function of Pol I, which normally hydrolizes the RNA primer, and then fills in the resulting single-stranded gap. To the Pol I enzyme, a PCR primer hybridized to a single- stranded region of DNA looks like a region of DNA that has just had the RNA primer removed.

I hope that this answers your question Anisha; you can find out even more about DNA replication by reviewing a good, college-level Biochemistry textbook, like Biochemistry by L. Stryer. You can find this text online, and I suggest reviewing Stryer 5th edition, Chapter 27.4.4 and Stryer, 5th Edition, Chapter 27.4.5.

Finally, I should add that the information I've provided above for Pol III pertains primarily to E. coli Pol III. You might want to read the following article for information about Pol III from an extreme thermophile (Aquifex aeolicus):
Irina Bruck, Alexander Yuzhakov, Olga Yurieva, David Jeruzalmi, Maija Skangalis, John Kuriyan, and Mike O’Donnell (2002)
Analysis of a Multicomponent Thermostable DNA Polymerase III Replicase from an Extreme Thermophile
Journal of Biological Chemistry 277(19) 17334–17348.

Keep asking questions!