A single side chain prevents Escherichia coli DNA polymerase I (Klenow fragment) from incorporating ribonucleotides

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520

Communicated by Thomas A. Steitz, Yale University, New Haven, CT (received for review December 25, 1997)

Abstract

Although nucleic acid polymerases from different families show striking similarities in structure, they maintain stringent specificity for the sugar structure of the incoming nucleoside triphosphate. The Klenow fragment of E. coli DNA polymerase I selects its natural substrates, deoxynucleotides, over ribonucleotides by several thousand fold. Analysis of mutant Klenow fragment derivatives indicates that discrimination is provided by the Glu-710 side chain which sterically blocks the 2′-OH of an incoming rNTP. A nearby aromatic side chain, at position 762, plays an important role in constraining the nucleotide so that the Glu-710 “steric gate” can be fully effective. Even with the E710A mutation, which is extremely permissive for addition of a single ribonucleotide to a DNA primer, Klenow fragment does not efficiently synthesize pure RNA, indicating that additional barriers prevent the incorporation of successive ribonucleotides.

Footnotes

↵ * Present address: Life Technologies, Inc., Rockville, MD 28050.
↵ † To whom reprint requests should be addressed at: Department of Molecular Biophysics and Biochemistry, Yale University, Bass Center for Molecular and Structural Biology, 266 Whitney Avenue, P.O. Box 208114, New Haven, CT 06520-8114. e-mail: catherine.joyce{at}yale.edu.
ABBREVIATIONS:

Kf pol,

Klenow fragment of DNA polymerase I;

ddNTP,

2′,3′-dideoxynucleoside triphosphate;

MMLV-RT,

Moloney murine leukemia virus reverse transcriptase