DNA Polymerase Fidelity Mechanisms

Abstract

DNA polymerase is a protein that catalyses the polymerization of one strand of DNA, called the primer strand, based on the sequence in another strand, called the template strand. Fidelity in this process refers to the ability of the polymerase to avoid or to correct errors in the newly synthesized strand.

Keywords: mutation; proofreading; exonuclease; processivity proteins; SOS response

Figure 1.

Exonuclease and polymerase active sites compete for binding of the primer terminus. (The exo site is on the left and the pol site is on the right, as drawn.) The primer 3′ end generally binds in the pol site, allowing polymerization to occur. Binding in the exonuclease active site is favoured when the primer strand separates from the template strand and presents itself as a short stretch of single‐stranded DNA. Separation of the two strands is more likely when base pairs are not correctly matched, biasing exonuclease activity towards excision of mismatches. The small circles represent the phosphates on the dNTPs and the released inorganic pyrophosphate following nucleotide incorporation.

Figure 2.

Misaligned primer/template structures can lead to mutation. This transient slippage phenomenon is most common in sequences with short, homopolymer repeats. When a base in the template strand rotates out of the helical plane, e.g. base T loops out, its original primer partner A can pair opposite a downstream template T. Continued synthesis from the 3′ end of this misaligned primer terminus, beginning with the incorporation of C opposite G, results in a −1 deletion mutation. Alternatively, a +1 addition mutation occurs if a primer base rotates out of the helical plane (not shown). Note that a base substitution mutation can also occur if the extrahelical template T rotates back into the helical plane, resulting in realignment of the DNA, causing the newly inserted C paired opposite G, to switch positions and become mispaired opposite T (not shown). Still another mechanism resulting in a −1 frameshift (or base substitution) occurs when a template base rotates transiently out of the plane of the DNA helix, and a dNTP bound at the polymerase active site pairs with the downstream template position (shown in the figure as dCTP paired opposite G, ‘dNTP‐stabilized’ template loop‐out structure). As before, continued synthesis on the misaligned structure leads to a −1 deletion, while realignment of the primer/template strands leads to a base substitution mutation.

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References

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Further Reading

Echols H and Goodman MF (1991) Fidelity mechanisms in DNA replication. Annual Review of Biochemistry 60: 477–511.

Fersht A (1985) Enzyme Structure and Mechanism. New York: WH Freeman.

Goodman MF (1995) DNA models: Mutations caught in the act. Nature 378: 237–238.

Goodman MF, Creighton S, Bloom LB and Petruska J (1993) Biochemical basis of DNA replication fidelity. Critical Reviews in Biochemistry and Molecular Biology 28: 83–126.

Woodgate R and Levine AS (1996) Damage inducible mutagenesis: Recent insights into the activities of the Umu family of mutagenesis proteins. Cancer Surveys 28: 117–140.

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Fygenson, Deborah Kuchnir, and Goodman, Myron F(Apr 2001) DNA Polymerase Fidelity Mechanisms. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0001053]