Proofreading function of DNA polymerase

A FIGURE 23-24 Schematic model of the proofreading function of DNA polymerases. All DNA polymerases have a similar three-dimensional structure, which resembles a half-opened right hand. The "fingers" bind the single-stranded segment of the template strand, and the polymerase catalytic activity (Pol) lies in the junction between the fingers and palm. 

Maintenance of the integrity of chromosomal DNA is essential for the survival of all forms of life. In part this is achieved through the proofreading function of DNA polymerases. However, errors in DNA polymerization occasionally escape this proofreading, and the sequence of bases in the DNA is altered by agents that are present in a cell or in its external environment. Both prokaryotes and enkaryotes have evolved elaborate DNA repair mechanisms to correct errors in the sequence of bases in DNA. 

The two primary functions of DNA polymerase are to read a template DNA strand and catalyze the polymerization of a new daughter strand, and to proofread the newly synthesized strand and correct any errors by removing the incorrectly inserted nucleotide and adding the proper one. 3 -TACGCCGATCTTATAAGGT-5 ... 

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 3 —>5 exonuclease activity of DNA polymerase I, at least, functions to proofread for such mistakes. After the incorrect base is incorporated, it will not remain hydrogen-bonded to the tautomeric base in the template once the latter returns, almost immediately, to its more stable form. The 3 — 5 exonuclease activity shows a strong preference for a frayed or non-hydrogen-bonded end and removes the misincorporated nucleotide before chain growth proceeds further. DNA polymerase III holoenzyme also has the potential to proofread by the same mechanism. 

Polymerase a was the first discovered, and it has the most subunits. It also has the ability to make primers, but it lacks a 3 5 proofreading activity and has low processivity. After making the RNA primer, Pol a adds about 20 nucleotides and is then replaced by Pol 5 and e. Polymerase 5 is the principal DNA polymerase in eukaryotes. It interacts with a special protein called PCNA ior proliferating cell nuclear antigen). PCNA is the eukaryotic equivalent of the part of Pol III that functions as a sliding clamp (P). It is a trimer of three identical proteins that surround the DNA (Figure 10.19). The role of DNA polymerase e is less clear. It may replace polymerase 5 in lagging strand synthesis. DNA polymerase p appears to be a repair enzyme. DNA polymerase y carries out DNA replication in mitochondria. Several... 

The majority of archaeal Family B DNA polymerase homologs function as monomers, lack 5 -> 3 -exonuclease activity, and possess an associated 3 5 -exonuclease (proofreading) activity. The editing capability of archaeal Family B DNA polymerases has led to their use in high-fidelity PCR applications. Sequence comparisons have identified three conserved motifs (exo I, II, IB) in the 3 -> 5 -exonuclease domain of DNA polymerases (reviewed ). Replacement of any of the conserved aspartic or glutamic acid residues with alanine has been shown to abolish the exonuclease activity of numerous DNA polymerases, including... 

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... 

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... 

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. 

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. 

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... 

PCR reactions should be performed using a thermostable DNA polymerase with proofreading function, i.e., Vent, New England Biolabs Beverly, MA, or Pfu, Stratagene, La Jolla, CA. The protocol used will vary with the source of target DNA and enzyme used. 

High-fidelity chromosomal replication in E. coli is executed by a multicomponent complex referred to as DNA polymerase III holoenzyme (see Fig. 4a) (18-21). Pol 111 holoenzyme consists of three main subcomponents Pol 111 core, 3-clamp, and y-complex clamp-loader. Pol 111 core is the replicative DNA polymerase that consists of three subunits (a, e, 0) a exhibits DNA polymerase activity, e performs 3 -5 exonuclease activity necessary for proofreading, and the function of 0 is currently unclear. 

Genetic engineered heat resistant DNA polymerases, that have proofreading functions and make fewer mutations in the amplified DNA products, are available commercially. PCR reactions are now carried out in different thermocyclers. Thermocyclers are designed to change temperatures automatically. Researchers set the temperatures and the time, and at the end of the procedure take the test tube out of the machine. 

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