Open AccessA single side chain prevents Escherichia coli DNA polymerase I (Klenow fragment) from incorporating ribonucleotides
Author(s) -
Mekbib Astatke,
Kimmie Ng,
Nigel D. F. Grindley,
Catherine M. Joyce
Publication year - 1998
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.95.7.3402
Subject(s) - klenow fragment , dna polymerase i , dna polymerase , polymerase , primer (cosmetics) , dna , dna clamp , primase , dna polymerase ii , chemistry , nucleic acid , biology , microbiology and biotechnology , biochemistry , stereochemistry , reverse transcriptase , rna , exonuclease , gene , organic chemistry
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 ofE. 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.