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. 1998 Mar 31;95(7):3507-12.
doi: 10.1073/pnas.95.7.3507.

Identification of a transient excision intermediate at the crossroads between DNA polymerase extension and proofreading pathways

R P Baker  1 , L J Reha-Krantz
Affiliations

Identification of a transient excision intermediate at the crossroads between DNA polymerase extension and proofreading pathways

R P Baker et al. Proc Natl Acad Sci U S A. .

Abstract

DNA polymerases achieve accurate DNA replication through a delicate balance between primer elongation and proofreading. While insufficient proofreading results in DNA replication errors, indiscriminate removal of correct along with incorrect nucleotides is wasteful and may prevent completion of DNA synthesis. The transition between polymerization and proofreading modes is proposed to be governed by a kinetic barrier that prevents proofreading unless the rate of primer elongation is significantly reduced by the presence of an incorrect base pair at the primer-terminus. We have used mutational analysis, coupled with a sensitive, fluorescence-based assay to characterize intermediate steps in the proofreading pathway. A highly fluorescent complex forms between the bacteriophage T4 DNA polymerase and DNA primer-templates labeled at the 3' terminus with the base analog 2-aminopurine. Formation of the fluorescent complex appears to be a rate-determining step in the proofreading pathway and is impaired for several mutator T4 DNA polymerases with amino acid substitutions in the exonuclease domain. Although these mutant DNA polymerases are proficient in hydrolysis, their reduced ability to form the fluorescent complex imposes a higher kinetic barrier. As a consequence, the mutant DNA polymerases proofread less frequently, resulting in more DNA replication errors.

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Figures

Figure 1
Figure 1
A minimal kinetic scheme for T4 DNA polymerase proofreading. P corresponds to 2AP and d2APMP is 2-aminopurine deoxynucleoside monophosphate.
Figure 2
Figure 2
Time courses for the formation of fluorescent complexes in the absence of Mg2+ for the wild-type and mutant T4 DNA polymerases with the G+C-rich 2AP-DNA substrate. A single-exponential curve fit is shown superimposed on the data for the wild-type and D131N-DNA polymerases. The time course for the G255S-DNA polymerase was better described by a double-exponential equation. Fluorescence intensity remained at the background level (equivalent to the fluorescence of a mixture of enzyme, DNA, EDTA, and DTT) in the time course with the D131G-DNA polymerase.
Figure 3
Figure 3
Exonucleolytic degradation of 32P-labeled d(T)16 by the wild-type and D131N-DNA polymerases. Reaction time points of 1, 5, 10, and 20 min are indicated above each lane. Full digestion is the dinucleotide product.
Figure 4
Figure 4
Magnified view of the T4 DNA polymerase exonuclease active center. Residues D112 and E114 reside within the exonuclease active center. Proposed hydrogen bonds between D131 and Y317 and between D131 and T116 are illustrated. The Y317 and E114 residues are separated by 4 Å and may interact indirectly through an ordered water molecule (J. Wang and T. A. Steitz, personal communication).
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References

    1. Johnson K A. Annu Rev Biochem. 1993;62:685–713. - PubMed
    1. Goodman M F, Creighton S, Bloom L B, Petruska J. Crit Rev Biochem Mol Biol. 1993;28:83–126. - PubMed
    1. Cowart M C, Gibson K J, Allen D J, Benkovic S J. Biochemistry. 1989;28:1975–1983. - PubMed
    1. Marquez L A, Reha-Krantz L J. J Biol Chem. 1996;271:28903–28911. - PubMed
    1. Ollis D L, Brick P, Hamlin R, Xuong N G, Steitz T A. Nature (London) 1985;313:762–766. - PubMed

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