Fidelity DNA polymerase

Mammals have many low-fidelity DNA polymerases of the TLS polymerase family. However, the presence of these enzymes does not necessarily translate into an unacceptable mutational burden, because most of the... 

L.G. Brieba et al., Structural basis for the dual coding potential of 8-oxoguanosine by a high-fidelity DNA polymerase. EMBO J. 23, 3452-3461 (2004)... 

Oxidative damage that leads to replication fork arrest is common during bacterial growth and probably occurs at least once during each cell division even under the most favorable conditions (48, 51). Most lesions are not dealt with by mutageiuc polymerases, but instead they are repaired by recombination, which is an error-free process. Recombinational repair can take many different paths and employs several different proteins, but ultimately, it involves high-fidelity DNA polymerases such as Pol I, Pol II, and/or Pol III (6, 49, 56). For additional information, we refer the reader to more thorough reviews of the various recombination repair pathways (44, 48-50,70). 

Even high-fidelity DNA polymerases sometimes incorporate an incorrect nucleotide. All cells contain a specialized mismatch repair system that catches these errors and corrects them. Mismatch repair involves excision of DNA past the mismatch, and eventually, it allows Pol III to try again (reviewed in References 71 and 72). The process of mismatch repair requires proteins MutS and MutL, among others. Mutations in the human homo-logues of these proteins lead to a predisposition to development of tumors (73). 

Indian C, Mclnerney P, Georgescu R, Goodman ME, O Donnell M. A sliding-clamp toolbelt binds high- and low-fidelity DNA polymerases simultaneously. Mol. Cell 2005 19 805-815. Bunting KA, Roe SM, Pearl LH. Structural basis for recruitment of translesion DNA polymerase Pol JV/DinB to the beta-clamp. Embo. J. 2003 22 5883-5892. 

It has been shown that 2,3-difluorotoluene (36) serves as a nonpolar shape mimic of thymine (37) and difluorotoluene deoxyriboside (38) is, surprisingly, an excellent nonpolar nucleoside isostere of thymidine (39) (see Fig. 1.21) [126, 127], Thus, the nucleotide of 36 was incorporated into DNA by several high-fidelity DNA polymerases [126, 128],... 

Kim, T. W., Brieba, L. G., Ellenberger, T. and Kool, E. T. (2006) Functional evidence for a small and rigid active site in a high fidelity DNA polymerase probing T7 DNA polymerase with variably sized base pairs. J. Biol. Chem., 281, 2289-2295. 

Hsu, G.W., Ober, M Carell, T and Beese, L.S. (2004) Error-prone replicabon of oxidabvely damaged DNA by a high-fidelity DNA polymerase. Nature, 431, 217-221. 

Hsu, G.W., Huang, X., Luneva, N.P., Geacintov, N.E., and Beese, LS. (2005) Structure of a high fidelity DNA polymerase bound to a benzo[o]pyrene adduct that blocks replication. 

The construction of chimeric enzyme genes was carried out using a self-priming PCR. High-fidelity DNA polymerase (KOD-Plus, Toyobo Biochemicals, Japan) was used in three-step PCR reactions to overcome incorporation of undesired errors during multiple PCR steps. 

Johnson KA (2010) The kinetic and chemical mechanism of high-fidelity DNA polymerases. Biochim Biophys Acta 1804(5) 1041-1048... 

Set up a PCR with PfuUltra II Fusion Hotstart DNA polymerase or Phusion Hotstart II High-Fidelity DNA polymerase using the product from the Taq ligation step as the template to amplify the 3-kb product. 

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