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

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.

Gln84 of moloney murine leukemia virus reverse transcriptase regulates the incorporation rates of ribonucleotides and deoxyribonucleotides

“…3). Similar pausing sites were also observed with other DNA polymerase mutants that acquired RNA polymerase activities [9,12,18,19]. One possible explanation is that for the enzyme to accommodate the RNA products containing 2-7 rNTPs, the active site is changed to a position that is not suitable for efficient catalysis.…”

Comparative Kinetics of Nucleotide Analog Incorporation by Vent DNA Polymerase

“…RB69 DNA polymerase also shows effects in both K D and k pol , achieving an even higher discrimination by virtue of a 230-fold weaker ground state binding. Discrimination against NTPs has, in large part, been attributed to a steric clash between the 2Ј-OH and a conserved side chain in the polymerase active site (26,30,32 b The kinetic parameters for Vent A488L DNA polymerase are from single determinations.…”

“…In the case of Family A DNA polymerases from bacteriophage T7, Escherichia coli (Klenow fragment, large fragment of DNA polymerase I), and Thermus aquaticus, as well as the Family B DNA polymerase from bacteriophage RB69, interpretation of the structural information is complemented by steady-state and pre-steady-state kinetic studies, allowing a detailed description of the polymerization pathway. Reaction parameters describing the discrimination against naturally occurring nucleotide analogs encountered in vivo, such as NTPs, or unnatural nucleotide analogs, such as ddNTPs and dyelabeled ddNTPs (13,(25)(26)(27)(28)(29)(30), have added insights into the basis for nucleotide discrimination.Hyperthermophilic archaeal DNA polymerases have not been scrutinized in such detail, hampering a complete characterization and comparison with other polymerases. Family B DNA polymerases from hyperthermophilic Archaea Thermococcus sp.…”

Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases

“…Early studies (reviewed in (1,2)) revealed that DNA polymerases do incorporate rNMPs during DNA synthesis. Kinetic studies (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) have further demonstrated that selectivity for insertion of dNMPs into DNA rather than rNMPs varies from 10-fold to >10 6 -fold, depending on the DNA polymerase and the dNTP/rNTP pair examined. rNMP incorporation during DNA synthesis is potentially made more probable by the fact that the concentrations of rNTPs in vivo are higher than are the concentrations of dNTPs (e.g., see refs.…”