DNA polymerase proofreading

The thermostable DNA polymerases can be divided into two groups those with a 3 5 exonuclease (proofreading) activity, such as Pfu DNA polymerase, and those without the proofreading function, such as Taq DNA polymerase. Both groups have some important differences. Proofreading DNA polymerases are significantly more accurate than nonproofreading polymerases, due to the 3 5 exonuclease activity,... 

A number of different DNA polymerase molecules engage in DNA replication. These share three important properties (1) chain elongation, (2) processivity, and (3) proofreading. Chain elongation accounts for the rate (in nucleotides per second) at which polymerization occurs. Processivity is an expression of the number of nucleotides added to the nascent chain before the polymerase disengages from the template. The proofreading function identifies copying errors and corrects them. In E coli, polymerase III (pol III) functions at the... 

Moser, M. J., Holley, W. R., Chatterjee, A., and Mian, I. S. (1997). The proofreading domain of Escherichia coli DNA polymerase land other DNA and/ or RNA exonuclease domains. Nucleic Acids Res. 25, 5110-5118. 

DNA polymerases can correct mistakes ( proofreading ), whereas RNA polymerases cannot. DNA polymerases have 3 —> 5 exonuclease activity for proofreading. 

When base selection and proofreading are combined, DNA polymerase leaves behind one net error for every 106 to 108 bases added. Yet the measured accuracy of replication in E. coli is higher still. The additional accuracy is provided by a separate enzyme system that repairs the mismatched base pairs remaining after replication. We describe this mismatch repair, along with other DNA repair processes, in Section 25.2. 

DNA polymerase I, then, is not the primary enzyme of replication instead it performs a host of clean-up functions during replication, recombination, and repair. The polymerase s special functions are enhanced by its 5 —>3 exonuclease activity. This activity, distinct from the 3 —>5 proofreading exonuclease (Fig. 25-7), is located in a structural domain that can be separated from the enzyme by mild protease treatment. When the 5 —>3 exonuclease domain is removed, the remaining fragment (Afr 68,000), the large fragment or Klenow fragment (Fig. 25-8), retains the polymerization and... 

DNA polymerase III is much more complex than DNA polymerase I, having ten types of subunits (Table 25-2). Its polymerization and proofreading activities reside in its a and e (epsilon) subunits, respectively. The 6 subunit associates with a and e to form a core polymerase, which can polymerize DNA but with limited processivity. Two core polymerases can be linked by... 

FIGURE 25-8 Large (Klenow) fragment of DNA polymerase I. This polymerase is widely distributed in bacteria. The Klenow fragment, produced by proteolytic treatment of the polymerase, retains the polymerization and proofreading activities of the enzyme. The Klenow fragment shown here is from the thermophilic bacterium Bacillus stearothermophilus (PDB ID 3BDP). The active site for addition of nucleotides is deep in the crevice at the far end of the bound DNA. The dark blue strand is the template. 

Like bacteria, eukaryotes have several types of DNA polymerases. Some have been linked to particular functions, such as the replication of mitochondrial DNA. The replication of nuclear chromosomes involves DNA polymerase a, in association with DNA polymerase S. DNA polymerase a is typically a multisubunit enzyme with similar structure and properties in all eukaryotic cells. One subunit has a primase activity, and the largest subunit (Afr -180,000) contains the polymerization activity. However, this polymerase has no proofreading 3 —>5 exonuclease activity, making it unsuitable for high-fidelity DNA replication. DNA polymerase a is believed to function only in the synthesis of short primers (containing either RNA or DNA) for Okazaki fragments on the lagging strand. These primers... 

The fidelity of DNA replication is maintained by (1) base selection by the polymerase, (2) a 3 —>5 proofreading exonuclease activity that is part of most DNA polymerases, and (3) specific repair systems for mismatches left behind after replication. 

Exonuclease activity enables DNA polymerase III to proofread the newly synthesized DNA strand. 

Exonuclease activity In addition to having the 5 —>3 po ) merase activity that synthesizes DNA, and the 3 ->5 exonucleas activity that proofreads the newly synthesized DNA chain lik DNA polymerase III, DNA polymerase I also has a 5 - 3 exon clease activity that is able to hydrolytically remove the RN primer. [Note These activities are exonucleases because the remove one nucleotide at a time from the end of the DNA chaii rather than cleaving it internally as do the endonucleases (Figui 29.18).] First, DNA polymerase I locates the space ("nick between the 3 -end of the DNA newly synthesized by DNA pol] merase III and the 5 -end of the adjacent RNA primer. Next, DN... 

DNA chain elongation is catalyzed by DNA polymerase III using 5 -deoxyribonucleoside triphosphates as substrates. The enzyme "proofreads" the newly synthesized DNA, removing terminal mismatched nucleotides with its 3 —>5 exonuclease activity. 

RNA primers are removed by DNA polymerase I using its 5 —>3 exonuclease activity. The resulting gaps are filled in by this enzyme which can also proofread. The final phosphodiester linkage is catalyzed by DNA ligase. 

There are at least five classes of eukaryotic DNA polymerases. Pol a is a multisubunit enzyme, one subunit of which performs the primase function. Pol a 5 ->3 polymerase activity adds a short piece of DNA to the RNA primer. Pol 8 completes DNA synthesis on the leading strand and elongates each lagging strand fragment, using 3 ->5 exonuclease activity to proofread the newly synthesized DNA. Pol p and pol e are involved in carrying out DNA "repair," and pol y replicates mitochondrial DNA. 

Exonuclease activities, proofreading, and editing. DNA polymerase I not only catalyzes the growth of DNA chains at the 3 end of a primer strand but also, at about a 10-fold slower rate, the hydrolytic removal of nucleotides from the 3 end (31- 5 exonuclease activity). The same enzyme also catalyzes hydrolytic removal of nucleotides from the 5 end of DNA chains. This latter 5 - 3 exonuclease activity, the DNA polymerase activity, and the 3 -5 exonuclease activity all arise from separate active sites in the protein. DNA polymerases II and III do not catalyze... 

Even with its proofreading activity, E. coli DNA polymerase III still exhibits a measurable rate of nucleotide misincorporation (about one mistake per 1010 nucleotides incorporated). Mutants of E. coli DNA polymerase III can be isolated that have a lower than normal rate of misincorporation. Why might such mutants, which can be said to have hy-peraccurate DNA replication, be evolutionarily unfavorable ... 

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