Structures of Ternary Complexes of Rat DNA Polymerase β, a DNA Template-Primer, and ddCTP

Pelletier, H.; Sawaya, M.R.; Kumar, Arun; Wilson, Samuel H.; Kraut, Joseph

doi:10.1126/science.7516580

Cited by 729 publications

(996 citation statements)

References 87 publications

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“…This was proven by X-ray crystallographical structure determination for DNA polymerase I from Escherichia coli (Beese & Steitz, 1991) and for the DNA polymerase b from rat (Pelletier et al, 1994). Threedimensional structures of other polymerases like the retroviral HIV-1 reverse transcriptase (Kohlstaedt et al, 1996;Jacobo-Molina et al, 1996), the bacteriophage T7 RNA polymerase (Sousa et al, 1993) and gp43, a bacteriophage DNA polymerase of the eukaryotic pol a family (Wang et al, 1997) have been analyzed without resolving the active site metals.…”

Section: Discussionmentioning

confidence: 99%

Aminoglycoside binding to the hammerhead ribozyme: a general model for the interaction of cationic antibiotics with RNA 1 1Edited by J. Karn

Hermann

Westhof

1998

Journal of Molecular Biology

178

164

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A variety of drugs inhibit biological key processes by binding to a speci®c RNA component. We focus here on the well-analysed hammerhead ribozyme RNA that is inhibited by aminoglycoside antibiotics, a process considered as a paradigm for studying drug/RNA interactions. With insight gained from molecular dynamics simulations of the ribozyme in the presence of Mg 2 identi®ed by crystallography and of aminoglycosides in solution, a general model for aminoglycoside binding to RNA is proposed. A striking structurally based complementarity between the charged ammonium groups of the aminoglycosides and the metal binding sites in the hammerhead was uncovered. Despite dynamical exibility of the aminoglycosides, several of the intramolecular distances between the charged ammonium groups of the drugs were found to be rather constant. Intramolecular ammonium distances of the aminoglycosides span ranges similar to the interionic distances between Mg 2 in the hammerhead. Successful docking of aminoglycosides to the hammerhead ribozyme could be achieved by positioning the ammonium groups at the sites occupied by Mg 2 . The covalently linked ammonium groups of the aminoglycosides are thus able to complement in space the negative electrostatic potential created by a three-dimensional RNA fold. Consequently, it is suggested that aminoglycoside-derived sugars could constitute a basic set of yardstick synthons ideal for rational and combinatorial synthesis of drugs targeted at biologically relevant RNA folds.

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Section: Discussionmentioning

confidence: 99%

Aminoglycoside binding to the hammerhead ribozyme: a general model for the interaction of cationic antibiotics with RNA 1 1Edited by J. Karn

Hermann

Westhof

1998

Journal of Molecular Biology

178

164

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“…Ca 2+ inhibits the ligase by binding to two sites, yet the ribozyme appears to be correctly folded. This raises the tantalizing possibility that the ligase uses a mechanism requiring two metal ions, similar to that of protein-enzyme polymerases (22,(53)(54)(55). In this mechanism, one metal ion coordinates the 3′-OH of the primer and the pro-R P -oxygen of the R-phosphate belonging to the incoming nucleotide (54), and the other contacts the nonbridging oxygens of the triphosphate, all through innersphere coordination (53)(54)(55).…”

Section: Role Of Metal Ions In Structure Andmentioning

confidence: 99%

“…The first metal ion promotes the attack of the 3′-OH on the R-phosphate by lowering its affinity for hydrogen, and the second stabilizes the pyrophosphate leaving group (22). This mechanism is common to several nonhomologous polymerases (22,(53)(54)(55), as well as to enzymes that catalyze other phosphoryl transfer reactions (albeit with differing stereochemical preferences), including the 3′-5′ exonuclease of DNA polymerase I and alkaline phosphatase (56)(57)(58)(59).…”

Section: Role Of Metal Ions In Structure Andmentioning

confidence: 99%

Metal Ion Requirements for Structure and Catalysis of an RNA Ligase Ribozyme

2002

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The class I ligase, a ribozyme previously isolated from random sequence, catalyzes a reaction similar to RNA polymerization, positioning its 5′-nucleotide via a Watson-Crick base pair, forming a 3′,5′-phosphodiester bond between its 5′-nucleotide and the substrate, and releasing pyrophosphate. Like most ribozymes, it requires metal ions for structure and catalysis. Here, we report the ionic requirements of this self-ligating ribozyme. 2+ and Co(NH 3 ) 6 3+ inhibit by binding at least two sites, but they appear to productively fill a subset of the required sites. Inhibition is not the result of a significant structural change, since the ribozyme assumes a nativelike structure when folded in the presence of Ca 2+ or Co(NH 3 ) 6 3+ , as observed by hydroxyl-radical mapping. As further support for a nativelike fold in Ca 2+ , ribozyme that has been prefolded in Ca 2+ can carry out the self-ligation very quickly upon the addition of Mg 2+ . Ligation rates of the prefolded ribozyme were directly measured and proceed at 800 min -1 at pH 9.0.Metal ions are essential for the activity of most ribozymes, functioning in electrostatic shielding, structure, and directly in catalysis (1). Natural ribozymes vary widely in their metal ion specificity, from the large ribozymes (group I intron, group II intron, and RNase P 1 ), which require a divalent metal, preferably Mg 2+ , for activity, to the promiscuous selfcleaving ribozymes (hammerhead, hairpin, VS, and HDV), which are active in many different cations (2-13). Hammerhead, hairpin, and VS ribozymes are even active in nonmetal ions such as NH 4 + (12, 13). Ribozymes selected in vitro also exhibit a wide range of metal ion requirements. Some, like the ribonucleolytic leadzyme, are active only in the cations in which they were selected (14-16). Others exhibit activity in the presence of metal ions which were not present during their selection. For example, an acyl transferase ribozyme, which was selected in the presence of Mg 2+ and K + only, is active in a wide variety of cations, including Co(NH 3 ) 6 3+ (17, 18).The class I ligase, which was also selected in the presence of only Mg 2+ and K + , performs a reaction similar to RNA polymerization, positioning its 5′-nucleotide via a WatsonCrick base pair, forming a 3′,5′-phosphodiester bond between its 5′-nucleotide and the substrate, and releasing pyrophosphate (Figure 1) (19). In fact, a version of this ribozyme can act as a general RNA polymerase, accurately extending a primer up to 14 nucleotides (20). Not only does the ligase perform the same reaction as naturally occurring RNA and DNA polymerases, but the stereospecificity of its preference for sulfur substitutions at the nonbridging oxygens on the R-phosphate matches that of these enzymes, raising the possibility that a catalytic mechanism common to all polymerases extends to ribozyme-mediated polymerization as well (21,22). The ligase is also one of the fastest ribozymes, among both natural ribozymes and those selected in vitro (23). These characteristics r...

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“…The structural and biochemical data thus suggest that Tyr-766 of Klenow fragment may have a function similar to that of Arg-283 in DNA polymerase ␤. In the crystal structure of the rat pol ␤⅐DNA⅐ddCTP ternary complex, the side chain of Arg-283 is in a position to interact with the template base in the polymerase active site (33). Replacement of arginine 283 with lysine, leucine, or alanine yields ␤ polymerases with strongly reduced catalytic efficiency and base substitution fidelity (34).…”

Section: Fig 1 Error Spectra For Klenow Polymerasesmentioning

confidence: 99%

Base Miscoding and Strand Misalignment Errors by Mutator Klenow Polymerases with Amino Acid Substitutions at Tyrosine 766 in the O Helix of the Fingers Subdomain

Bell

Eckert

Joyce

et al. 1997

Journal of Biological Chemistry

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A mutant derivative of Klenow fragment DNA polymerase containing serine substituted for tyrosine at residue 766 has been shown by kinetic analysis to have an increased misinsertion rate relative to wild-type Klenow fragment, but a decreased rate of extension from the resulting mispairs (Carroll, S. S., Cowart, M., and Benkovic, S. J. (1991) Biochemistry 30, 804 -813). In the present study we use an M13mp2-based fidelity assay to study the error specificity of this mutator polymerase. Despite its compromised ability to extend mispairs, the Y766S polymerase and a Y766A mutant both have elevated base substitution error rates. The magnitude of the mutator effect is mispair-specific, from no effect for some mispairs to rates elevated by 60-fold for misincorporation of TMP opposite template G. The results with the Y766S mutant are remarkably consistent with the earlier kinetic analysis of misinsertion, demonstrating that either approach can be used to identify and characterize mutator polymerases. Both the Y766S and Y766A mutant polymerases are also frameshift mutators, having elevated rates for two-base deletions and a 276-base deletion between a direct repeat sequence. However, neither mutant polymerase has an increased error rate for single-base frameshifts in repetitive sequences. This error specificity suggests that the deletions generated by the mutator polymerases are initiated by misinsertion rather than by strand slippage. When considered with recent structure-function studies of other polymerases, the data indicate that the nucleotide misinsertion and strand-slippage mechanisms for polymerization infidelity are differentially affected by changes in distinct structural elements of DNA polymerases that share similar subdomain structures.Among the most important properties of a DNA polymerization reaction is its fidelity. Numerous studies (reviewed in Refs. 1-3) have shown that two events initiate most polymerization errors. One is the misinsertion of an incorrect nucleotide. This usually yields a base substitution mutation, but can also yield a frameshift when the misinserted nucleotide is complementary to an adjacent template base and the primer relocates to produce misaligned strands. Several steps in the reaction cycle can affect the rate of errors initiated by misinsertion, including dNTP binding, a subsequent conformational change preceding chemistry and the rate of phosphodiester bond formation. Also critical is the balance between extension of a misinserted base and its exonucleolytic removal or rearrangement. The second error-initiating event is template-primer slippage (Ref. 4, reviewed in Ref. 2), usually resulting in deletion or addition of one or more nucleotides, particularly in repetitive sequences.One approach for understanding these two error-initiating events is to study the properties of DNA polymerases whose x-ray crystal structures have revealed interactions with the template-primer and incoming dNTPs that might influence fidelity. Studies of four polymerases (reviewed in Ref. 5) indicate ...

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Structures of Ternary Complexes of Rat DNA Polymerase β, a DNA Template-Primer, and ddCTP

Cited by 729 publications

References 87 publications

Aminoglycoside binding to the hammerhead ribozyme: a general model for the interaction of cationic antibiotics with RNA 1 1Edited by J. Karn

Aminoglycoside binding to the hammerhead ribozyme: a general model for the interaction of cationic antibiotics with RNA 1 1Edited by J. Karn

Metal Ion Requirements for Structure and Catalysis of an RNA Ligase Ribozyme

Base Miscoding and Strand Misalignment Errors by Mutator Klenow Polymerases with Amino Acid Substitutions at Tyrosine 766 in the O Helix of the Fingers Subdomain

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