3 —>5 proofreading exonuclease

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... 

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. 

Pol/3, like Pola and PolS is also located in the nucleus and is probably involved in DNA repair. Poly is localized in mitochondria, and although there is no direct evidence, it is thought to be responsible for the replication of that organelle s DNA. Pol5 has 3 — 5 proofreading exonuclease action that is highly selective for base mismatches. 

The 3 -5 proofreading exonuclease is called into action when an incorrect dNMP is added to the 3 terminus. The 3 -5 exonuclease is located in a separate domain with a distinct active site. The chemical reaction of the exonuclease proceeds by hydrolysis (see Fig. lb), but it is remarkably similar to the polymerase reaction. Specifically, two metal ions catalyze the reaction metal ion A activates water to form a hydroxyanion nucleophile that attacks the phosphodiester bond of the 3 terminal mismatched nucleotide. Metal B stabilizes the developing charge on the dNMP leaving group. 

DNA polymerase III is an asymmetric dimer. It contains two copies of the core polymerase, snbnnits a, e, and 0. The a subunit has polymerase activity while e is a 3 — 5 proofreading exonuclease. A 2 subunit is associated with one arm and a (55 i/)2 subunit with the other. These serve as the clamp-loading complex. The P2 subunits form ringlike structures that serve as sliding DNA clamps. 

DNA polymerase 7 is a member of family A and is the only cellular polymerase known to be present in mitochondria (Kaguni, 2004). Thus, it is believed to be responsible for replication of mitochondrial DNA, as well as for any repair that occurs in mitochondria (e.g., BER see above). Human Pol 7 is an accurate enzyme (Table II) and has an intrinsic 3 —> 5 proofreading exonuclease. It forms a tight complex with the p55 accessory subunit (Garrodeguas et al., 2001 Lim et al, 1999), which increases DNA binding affinity, stimulates the polymerase and exonuclease activities, and increases processivity. 

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