Fidelity of DNA polymerase

Linn, S Kairis, M. and Holliday, R. Decreased Fidelity of DNA Polymerase Activity Isolated from Aging Human Fibroblasts , Proc. Natl, Acad. Sci. (1976) 73, 2818. 

As well as alkylation or arylation of bases in DNA, intercalation of planar molecules within the double helix may also be important if it allows a subsequent reaction to take place. Also, the effect of carcinogens, such as some metals, on the fidelity of DNA polymerase may be another mechanism whereby altered DNA is produced. Some carcinogens, such as hydrazine, for example, can indirectly cause DNA methylation which may be involved in the carcinogenic process. After the initial reaction with DNA has taken place the damage is either repaired or fixed if replication takes place before repair. 

Florian, J., Goodman, M.F., and Warshel, A. (2005) Computer simulations of protein functions searching for the molecular origin of the replication fidelity of DNA polymerases. Proc. Natl. Acad. Sci. USA, 102, 6819-6824. 

Neoplasms can undergo permanent stable changes in their phenotype, a process referred to as progression (33). Little is known about the basis for this alteration in the characteristics of neoplasms. It could result from gene amplification or a change in their chromosomal complement. Another hypothesis (34) is that decreased fidelity of DNA polymerases in tumor cells leads to errors in the replication of DNA, thereby introducing new mutations. 

Fidelity of DNA polymerases relative to the estimated value(s) of - 10 errors/bp/cycle for Ta DNA polymerase under usual PCR conditions. 

Pandey VN, Kaushik N, Rege N, Sarafianos SG, Yadav NS, Modak MJ. Role of M184 of human immunodeficiency virus type-1 reverse transcriptase in the polymerase function and fidelity of DNA synthesis. Biochem 1996 35 2168-2179. 

FIGURE 25-6 Contribution of base-pair geometry to the fidelity of DNA replication, (a) The standard A=T and G=C base pairs have very similar geometries, and an active site sized to fit one (blue shading) will generally accommodate the other, (b) The geometry of incorrectly paired bases can exclude them from the active site, as occurs on DNA polymerase I. 

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. 

Carroll, S. S., Cowaet, M., and Benkovic, S. J. (1991). A mutant of DNA polymerase I (Klenow Fragment) with reduced fidelity. Biochemistry 30, 804-813. 

Dong, Q., Copeland, W. C., and Wang, T. S. (1993). Mutational studies of human DNA polymerase a Identification of residues critical for deoxynucleotide binding and misinsertion fidelity of DNA synthesis. / Biol. Chem. 268, 24163-24174. 

Valuable insights into how DNA polymerases process their substrates were obtained as a result of detailed kinetic studies of the enzymes. Benkovic and coworkers employed rapid quenching techniques to study the kinetics of transient intermediates in the reaction pathway of DNA polymerases [5]. Intensive studies revealed that E. coli DNA polymerase I follows an ordered sequential reaction pathway when promoting DNA synthesis. Important aspects of these results for DNA polymerase fidelity are conformational changes before and after the chemical step and the occurrence of different rate-limiting steps for insertion of canonical and non-canonical nucleotides. E. coli DNA polymerase I discriminates between canonical and non-canonical nucleotide insertion by formation of the chemical bond. Bond formation proceeds at a rate more than several thousand times slower when an incorrect dNTP is processed compared with canonical nucleotide insertion. 

Taking together, DNA polymerase structural data indicate a high degree of shape complementary between the active sites of the enzymes and the nucleotide substrates, suggesting that geometrical constraints are at least one cause of DNA polymerase fidelity. 

A major goal of directed evolution of DNA polymerases has been to elucidate the structural elements that confer high fidelity during DNA replication. If DNA polymerases were to rely solely on the stability of nucleotides that aligned with template for discrimination of correct template-directed polymerization, the error frequency would be in the order of one mispaired nucleotide per 100 incorporated [23], The measured error rate for incorporation and extension of a mismatched nucleotide attributable to DNA polymerases lacking an error correcting exonucleolytic activity range... 

The fidelity of polymerization depends on the ability of the polymerase active site to discriminate complementary from non-complementary incoming nucleotide triphosphates as well as to extend matched from unmatched primer termini. Taq DNA polymerase does not contain a 3 -5 exonudease or proofreading activity. This makes it advantageous for studying the intrinsic fidelity of the polymerase active site for discrimination between complementary versus non-complementary base pairing. 

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