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A more recent version of this article appeared on September 6, 2002
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M204826200v1
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Papers In Press, published online ahead of print July 3, 2002
J. Biol. Chem, 10.1074/jbc.M204826200
Submitted on May 16, 2002
Revised on July 1, 2002
Accepted on July 2, 2002

Fidelity of Escherichia coli DNA polymerase IV: preferential generation of small deletion mutations By dNTP-stabilized misalignment

Sawami Kobayashi, Michael R. Valentine, Phuong Pham, Mike O'Donnell, and Myron F. Goodman

Biological Sciences and Chemistry, University of Southern California, Los Angeles, CA 90089-1340

Corresponding Author: mgoodman{at}mizar.usc.edu

Escherichia coli DNA polymerase IV (pol IV), a member of the error-prone Y-family, predominantly generates –1 frameshifts when copying DNA in vitro. T to G transversions and T to C transitions are the most frequent base substitutions observed. The in vitro data agree mutational spectra obtained when pol IV is overexpressed in vivo. Single base deletion and base substitution rates measured in the lacZa gene in vitro are, on average, 2 x 10-4 and 5 x 10-5, respectively. The range of misincorporation and mismatch extension efficiencies determined kinetically are 10-3 to 10-5. The presence of beta sliding clamp and gamma complex clamp loading proteins strongly enhance pol IV processivity, but have no discernible influence on fidelity. By analyzing changes in fluorescence of a 2-aminopurine template base undergoing replication in real-time, we show that a "dNTP-stabilized" misalignment mechanism is responsible for making –1 frameshift mutations on undamaged DNA. In this mechanism, a dNTP substrate is paired "correctly" opposite a downstream template base, on a "looped out" template strand instead of mispairing opposite a next available template base. Using the same mechanism, pol IV "skips" past an abasic template lesion to generate a –1 frameshift. A crystal structure depicting dNTP-stabilized misalignment was reported recently for Sulfolubus solfataricus Dpo4, a Y-family homolog of E. coli pol IV.


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