Enzyme proofreading

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. 

The hepatitis B virus (HBV) genome is one of the smallest viral genomes (approximately 3,200 base pairs) and encodes only one viral enzyme, namely the HBV reverse transcriptase (RT). Like the HIV RT, the HBV RT is an error-prone enzyme lacking proofreading activity. In combination with a high virus production, this results in an HBV quasispecies. 

Polymerase II (pol II) is mostly involved in proofreading and DNA repair. Polymerase I (pol I) completes chain synthesis between Okazaki fragments on the lagging strand. Eukaryotic cells have counterparts for each of these enzymes plus some additional ones. A comparison is shown in Table 36—6. 

Answer E. The 3 to 5 exonuclease activity of DNA pol 8 represents the proofreading activity of an enzyme required for the replication of human chromosomal DNA. DNA pol y (mitochondrial) and DNA pol III (prokaryotic) do not participate in this process, short RNA primers are replaced with DNA during replication, and new DNA strands are always... 

Fidelity of match between the template and the newly synthesized copy is maintained at a high level by enzymes with proofreading activity. 

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... 

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... 

Amino acids are activated by specific aminoacyl-tRNA synthetases in the cytosol. These enzymes catalyze the formation of aminoacyl-tRNAs, with simultaneous cleavage of ATP to AMP and PPj. The fidelity of protein synthesis depends on the accuracy of this reaction, and some of these enzymes carry out proofreading steps at separate active sites. In bacteria, the initiating aminoacyl-tRNA in all proteins is A-formylmethionyl-tRNAfMet. 

Enzymes that catalyze DNA chain elongation, proofreading, removal of RNA primers, filling gaps, and making the final phosphodiester bond... 

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. 

This editing mechanism for isoleucyl-tRNA synthetase was demonstrated directly in 1998 by X-ray crystallography on complexes of the enzyme with L-isoleucine and L-valine. Both substrates fit into the ATP-requir-ing synthetic site but neither isoleucine nor isoleucyl-tRNA will fit into the editing site which is located in an adjacent (3-barrel domain.104 105 Proofreading steps based on differing chemical properties as well as size can also be visualized.103 106... 

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... 

The N-terminal domain contains the 3, 5 -exonuclease activity, which is thought to fulfill a proofreading and editing function.2753 The polymerase acts at the 3 end of the growing DNA chain. Before moving on to the next position, the enzyme verifies that the correct base pair has been formed in the preceding polymerization event. If it has not, the exonuclease action removes the incorrect nucleotide and allows the polymerase to add the correct one. Thus, each base pair is checked... 

J. J. Hopfield has suggested a general mechanism called kinetic proofreading in which there is no hydrolytic site on the enzyme instead, the desired intermediates diffuse into solution, where they hydrolyze nonenzymatically.54 An example is in the selection of amino acids by the aminoacyl-tRNA synthetases (equation 13.32). 

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