Polymerase proofreading

Many Polymerases Proofread the Newly Added Bases and Excise Errors... 

Many DNA polymerases proofread the nascent product their 3 —> 5 exonuclease activity potentially edits the outcome of each polymerization step. A mispaired nucleotide is excised before the next step proceeds. In E. coli, DNA polymerase I repairs DNA and participates in replication. Fidelity is further enhanced by an induced fit that results in a catalytically active conformation only when the complex of enzyme, DNA, and correct dNTP is formed. Helicases prepare the way for DNA replication by using ATP hydrolysis to separate the strands of the double helix. 

The cellular DNA and RNA polymerases of higher organisms are much more accurate than those of viruses because of their high specificities and, in the case of DNA polymerases, proofreading capabilities. This accounts for the high degree of mutation in viruses. However, this can have therapeutic advantages. 

Fig. 3. sue with multiple inserts. A schematic iliustrating the assembiy of five inserts into a vector in a singie step. The vector and inserts are prepared by T4 DNA polymerase proofreading excision to reveal the homologous overhangs, annealed in an equimolar ratio, and transformed into cells. 

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... 

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. 

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. 

RNA polymerase moves along the template strand in the 3 to 5 direction as it synthesizes the RNA product in the 5 to 3 direction using NTPs (ATP, GTP, CTP, UTP) as substrates. RNA polymerase does not proofread its work. The RNA product is complementary and antiparallel to the template strand. 

Fig. 5. Illustration of the 5 -nucleotidase (TaqMan) assay for allele discrimination. (A) The allele discrimination assay employs two unlabeled PCR primers and two doubly fluorescent labeled PCR probes for visuaUzation of a mutant allele. The target sequence is initially denatured and amplified in the presence of each of the primers and probes. Increasing polymerization in the presence of a thermostable polymerase which contains a 5 proofreading function allows cleavage of one fluorescent indicator from an appropriate probe during the cycling reaction. (B) Probes are designed with a fluorescent reporter and a quencher moiety. AmpUfication reactions are spiked with additional fluorescent quenchers in order to render the reaction initially dark to the photomultipUer mbe or diode. The probes are designed...

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. 

RNA replicase isolated from Qj8-infected E. coli cells catalyzes the formation of an RNA complementary to the viral RNA, in a reaction equivalent to that catalyzed by DNA-dependent RNA polymerases. New RNA strand synthesis proceeds in the 5 —>3 direction by a chemical mechanism identical to that used in all other nucleic acid synthetic reactions that require a template. RNA replicase requires RNA as its template and will not function with DNA. It lacks a separate proofreading endonuclease activity and has an error rate similar to that of RNA polymerase. Unlike the DNA and RNA polymerases, RNA replicases are specific for the RNA of their own virus the RNAs of the host cell are generally not replicated. This explains how RNA viruses are preferentially replicated in the host cell, which contains many other types of RNA. 

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... 

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