Probing the active site tightness of DNA polymerase in subangstrom increments.

Abstract:

:We describe the use of a series of gradually expanded thymine nucleobase analogs in probing steric effects in DNA polymerase efficiency and fidelity. In these nonpolar compounds, the base size was increased incrementally over a 1.0-A range by use of variably sized atoms (H, F, Cl, Br, and I) to replace the oxygen molecules of thymine. Kinetics studies with DNA Pol I (Klenow fragment, exonuclease-deficient) in vitro showed that replication efficiency opposite adenine increased through the series, reaching a peak at the chlorinated compound. Efficiency then dropped markedly as a steric tightness limit was apparently reached. Importantly, fidelity also followed this trend, with the fidelity maximum at dichlorotoluene, the largest compound that fits without apparent repulsion. The fidelity at this point approached that of wild-type thymine. Surprisingly, the maximum fidelity and efficiency was found at a base pair size significantly larger than the natural size. Parallel bypass and mutagenesis experiments were then carried out in vivo with a bacterial assay for replication. The cellular results were virtually the same as those seen in solution. The results provide direct evidence for the importance of a tight steric fit on DNA replication fidelity. In addition, the results suggest that even high-fidelity replicative enzymes have more steric room than necessary, possibly to allow for an evolutionarily advantageous mutation rate.

authors

Kim TW,Delaney JC,Essigmann JM,Kool ET

doi

10.1073/pnas.0505113102

keywords:

subject

Has Abstract

pub_date

2005-11-01 00:00:00

pages

15803-8

issue

44

eissn

0027-8424

issn

1091-6490

pii

0505113102

journal_volume

102

pub_type

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