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

AUTOR(ES)

Astatke, Mekbib

FONTE

The National Academy of Sciences

RESUMO

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.

Documentos Relacionados

• Construction of a plasmid that overproduces the large proteolytic fragment (Klenow fragment) of DNA polymerase I of Escherichia coli.
• DNA Sequence Effects on Single Base Deletions Arising during DNA Polymerization in Vitro by Escherichia Coli Klenow Fragment Polymerase
• Mechanism of the idling-turnover reaction of the large (Klenow) fragment of Escherichia coli DNA polymerase I.
• Labelling DNA ends with the Klenow fragment of the E. coli DNA polymerase I: a cautionary note.
• Translesional synthesis on a DNA template containing N2-methyl-2′-deoxyguanosine catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I