Structural basis for the 3′-5′ exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism.

Beese, L.S.; Steitz, Thomas A.

doi:10.1002/j.1460-2075.1991.tb07917.x

Cited by 1,014 publications

(804 citation statements)

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“…Concomitantly, the bond between this phosphorus atom and the oxygen bridging the α and β-phosphates is broken, resulting in the release of pyrophosphate. By analogy to the mechanism proposed for a 3′ exonuclease reaction [4], a mechanism for this reaction was proposed in 1993 [5] and remains current today. DNA polymerases were proposed to catalyze the reaction with the help of two divalent metal ions, which in vivo are generally believed to be Mg 2+ , but sometimes may possibly be Mn 2+ [6].…”

Section: Introductionmentioning

confidence: 92%

Role of the catalytic metal during polymerization by DNA polymerase lambda

et al. 2007

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The incorporation of dNMPs into DNA by polymerases involves a phosphoryl transfer reaction hypothesized to require two divalent metal ions. Here we investigate this hypothesis using as a model human DNA polymerase λ (Pol λ), an enzyme suggested to be activated in vivo by manganese. We report the crystal structures of four complexes of human Pol λ. In a 1.9Å structure of Pol λ containing a 3′ OH and the non-hydrolyzable analog dUpnpp, a non-catalytic Na + ion occupies the site for metal A and the ribose of the primer-terminal nucleotide is found in a conformation that positions the acceptor 3'OH out of line with the α-phosphate and the bridging oxygen of the pyrophosphate leaving group. Soaking this crystal in MnCl 2 yielded a 2.0Å structure with Mn 2+ occupying the site for metal A. In the presence of Mn 2+ , the conformation of the ribose is C3' endo and the 3'-oxygen is in line with the leaving oxygen, at a distance from the phosphorus atom of the α-phosphate (3.69 Å) consistent with and supporting a catalytic mechanism involving two divalent metal ions. Finally, soaking with MnCl 2 converted a pre-catalytic Pol λ/Na + complex with unreacted dCTP in the active site into a product complex via catalysis in the crystal. These data provide pre-and post-transition state information and outline in a single crystal the pathway for the phosphoryl transfer reaction carried out by DNA polymerases.

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Section: Introductionmentioning

confidence: 92%

Role of the catalytic metal during polymerization by DNA polymerase lambda

et al. 2007

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“…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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“…However, there are subtle differences in the mode of DNA recognition and interaction displayed by these repressors, the details of which will be discussed later. On the other hand, the three helix-turn-helix repressors listed in Table 1 Beese & Steitz (1991 Beese & Steitz (1991) Freemont et al (1988) Steitz (1990) White et al (1989) Sanderson et al (1990 Burlingame et al (1985) Richmond et al (1984 is involved in the regulation of tryptophan biosynthesis by binding as a dimer to three different operator sites in the presence of its corepressor L-tryptophan (Klig et al, 1988). Binding in the absence of L-tryptophan is weak and non-specific.…”

Section: Helix-turn-helix Proteinsmentioning

confidence: 99%

“…Limited proteolysis cleaves the molecule into two fragments; the larger C-terminal domain (Klenow fragment, 605 amino acids) has both the DNA polymerase and 3'-5' exonuclease activities, whereas the smaller Nterminal domain has the 5'-3' exonuclease (for a recent review see Joyce, 1991). The crystal structure of the Klenow fragment complexed with thymidine monophosphate (dTMP) has been solved and shows that the protein is folded into two distinct domains (Ollis et al, 1985;Beese & Steitz, 1991). The larger Cterminal domain forms a deep cleft, the bottom of which is made up by a six-stranded antiparallel fl-sheet structure.…”

Section: Beta Proteinsmentioning

confidence: 99%