Side chains involved in catalysis of the polymerase reaction of DNA polymerase I from Escherichia coli.

Polesky, Andrea; Dahlberg, Michael E.; Benkovic, Stephen J.; Grindley, Nigel D. F.; Joyce, Catherine M.

doi:10.1016/s0021-9258(18)42461-1

Cited by 186 publications

(98 citation statements)

References 75 publications

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“…In contrast, in the presence of Stl, the rate of incorporation of α-thio-NTP (as compared to that of the NTP) was decreased by only 20-fold. Barring the potential complications from steric effects of Stl binding, these results suggest that the chemical step is no longer rate limiting in the presence of the inhibitor (Kaushik et al, 1996;Polesky et al, 1992). These results are consistent with our structural data, which show that the RNAP active groups (catalytic Asps and Mg 2+ ion) remain unaltered in the RNAP-Stl complex structure, as compared to that of free RNAP.…”

Section: Stl Does Not Target the Phosphodiester Bond Formationsupporting

confidence: 90%

“…Although Stl is positioned far away from the RNAP active site Asp residues, it could modulate catalysis allosterically. To investigate the effect of Stl on the phosphodiester bond formation, we used a slow-hydrolysable NTP analog, α-thio-NTP, which is thought to decrease the efficiency of nucleophilic attack by the nascent RNA's 3# OH due to the lesser electronegativity and a larger size of sulfur compared to oxygen (Kaushik et al, 1996;Polesky et al, 1992). We found that at conditions when RNAP was saturated with either α-thio-NTP or the corresponding NTP, α-thio-NTP was incorporated more than three orders of magnitude slower than the NTP (Table 2).…”

Section: Stl Does Not Target the Phosphodiester Bond Formationmentioning

confidence: 99%

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Structural Basis of Transcription Inhibition by Antibiotic Streptolydigin

et al. 2005

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Streptolydigin (Stl) is a potent inhibitor of bacterial RNA polymerases (RNAPs). The 2.4 A resolution structure of the Thermus thermophilus RNAP-Stl complex showed that, in full agreement with the available genetic data, the inhibitor binding site is located 20 A away from the RNAP active site and encompasses the bridge helix and the trigger loop, two elements that are considered to be crucial for RNAP catalytic center function. Structure-based biochemical experiments revealed additional determinants of Stl binding and demonstrated that Stl does not affect NTP substrate binding, DNA translocation, and phosphodiester bond formation. The RNAP-Stl complex structure, its comparison with the closely related substrate bound eukaryotic transcription elongation complexes, and biochemical analysis suggest an inhibitory mechanism in which Stl stabilizes catalytically inactive (preinsertion) substrate bound transcription intermediate, thereby blocking structural isomerization of RNAP to an active configuration. The results provide a basis for a design of new antibiotics utilizing the Stl-like mechanism.

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Section: Stl Does Not Target the Phosphodiester Bond Formationsupporting

confidence: 90%

Section: Stl Does Not Target the Phosphodiester Bond Formationmentioning

confidence: 99%

Structural Basis of Transcription Inhibition by Antibiotic Streptolydigin

et al. 2005

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“…In the crystal structures of T7 DNA polymerases complexed with various substrates, two Mg 2+ ions are invariably 4 Å apart in the ''closed'' active site (Doublie et al, 1998;Kim et al, 2005;Li et al, 2004), but one of the three catalytically essential carboxylates (E655) is not in position to coordinate the metal ions (Figure 3A). Substitution of the equivalent Glu in E. coli DNA polymerase I (E883) leads to normal metal ion and substrate binding but a reduced catalytic rate (Gangurde and Modak, 2002;Polesky et al, 1992). Engaging the third carboxylate in metal ion coordination may be the ratelimiting step.…”

Section: Advantages Of Two-metal-ion Catalysis: Specificity and Versatilitymentioning

confidence: 99%

Making and Breaking Nucleic Acids: Two-Mg2+-Ion Catalysis and Substrate Specificity

2006

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DNA and a large proportion of RNA are antiparallel duplexes composed of an unvarying phosphosugar backbone surrounding uniformly stacked and highly similar base pairs. How do the myriad of enzymes (including ribozymes) that perform catalysis on nucleic acids achieve exquisite structure or sequence specificity? In all DNA and RNA polymerases and many nucleases and transposases, two Mg2+ ions are jointly coordinated by the nucleic acid substrate and catalytic residues of the enzyme. Based on the exquisite sensitivity of Mg2+ ions to the ligand geometry and electrostatic environment, we propose that two-metal-ion catalysis greatly enhances substrate recognition and catalytic specificity.

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“…4, gold color). Among these conserved residues are the nine amino acids that coordinate with the DNA phosphate backbone in the KF-DNA editing complex structure [11] and the ten residues shown by mutagenesis to have an effect on substrate binding or catalysis [5,6]. The longest semi-continuous stretch of sequence identity in the polymerase domain includes residues 611-666 in BF (residues 664-718 in KF and 569-623 in Taq), which includes helices J-L and sheets 8 and 9.…”

Section: Polymerase Domainmentioning

confidence: 99%

“…The crystal structure of KF was determined at 3.3 Å resolution [3] and refined to 2.6 Å resolution [4]. Extensive site-directed mutagenesis, biochemical, and crystallographic studies of KF have provided a wealth of information characterizing the activity, mechanism, and structure of this DNA polymerase [3][4][5][6][7][8][9][10][11][12].…”

Section: Introductionmentioning

confidence: 99%

Crystal structure of a thermostable Bacillus DNA polymerase l large fragment at 2.1 Å resolution

Kiefer

Chen

Hansen³

et al. 1997

Structure

151

149

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This structure represents the highest resolution view of a Pol I enzyme obtained to date. Comparison of the three Pol I structures reveals no compelling evidence for many of the specific interactions that have been proposed to induce thermostability, but suggests that thermostability arises from innumerable small changes distributed throughout the protein structure. The polymerase domain is highly conserved in all three proteins. The N-terminal domains are highly divergent in sequence, but retain a common fold. When present, the 3'-5' proofreading exonuclease activity is associated with this domain. Its absence is associated with changes in catalytic residues that coordinate the divalent ions required for activity and in loops connecting homologous secondary structural elements. In BF, these changes result in a blockage of the DNA-binding cleft.

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Side chains involved in catalysis of the polymerase reaction of DNA polymerase I from Escherichia coli.

Cited by 186 publications

References 75 publications

Structural Basis of Transcription Inhibition by Antibiotic Streptolydigin

Structural Basis of Transcription Inhibition by Antibiotic Streptolydigin

Making and Breaking Nucleic Acids: Two-Mg2+-Ion Catalysis and Substrate Specificity

Crystal structure of a thermostable Bacillus DNA polymerase l large fragment at 2.1 Å resolution

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