Magnesium-Induced Assembly of a Complete DNA Polymerase Catalytic Complex

Batra, V.K.; Beard, William A.; Shock, David D.; Krahn, J.M.; Pedersen, Lars C.; Wilson, Samuel H.

doi:10.1016/j.str.2006.01.011

Cited by 243 publications

(459 citation statements)

References 48 publications

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“…The new structural data confirm the catalytic pathway predicted by earlier structures and stress the important role of the catalytic metal in inducing the ribose of the primer-terminal nucleotide to achieve an active conformation. The present structures are consistent with the depiction of the reaction mechanism described in Batra et al [8] for Pol β, an enzyme of the same family. In fact, an overlay of the Pol λ and Pol β complexes with dUpnpp (Fig.…”

Section: Discussionsupporting

confidence: 92%

“…Previous reports with Pol β suggest that Na + ions could out compete the Mg 2+ ion for the catalytic metal binding site [8]. When the above Pol λ crystals were soaked in a solution containing a higher MgCl 2 concentration, no difference was observed in the structure as a result of the soak (not shown).…”

Section: Mn 2+ As a Metal Activatormentioning

confidence: 64%

“…In fact, the 3'-oxygen is often missing because a favorite method to trap pre-catalytic complexes of DNA polymerases is to employ a dideoxy-terminated primer (i.e., missing the 3'-OH). The exception is a recent study by Wilson and colleagues [8] describing the structure of a pre-catalytic complex of DNA polymerase β bound to gapped DNA, with a non-hydrolyzable nucleotide analog and two divalent metal ions bound at the active site. This structure (discussed further below), and the present study of DNA polymerase λ, strongly support the proposal that two divalent metal ions are required for catalysis.…”

Section: Introductionmentioning

confidence: 99%

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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: Discussionsupporting

confidence: 92%

Section: Mn 2+ As a Metal Activatormentioning

confidence: 64%

Section: Introductionmentioning

confidence: 99%

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Role of the catalytic metal during polymerization by DNA polymerase lambda

et al. 2007

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“…The conformations of many of these enzyme side chains are also similar in these two binary complex structures. Asp-192 coordinates both active site Mg 2ϩ ions in the ''active'' ternary substrate complex (36). However, in the inactive binary DNA complex with unadducted or B[c]Ph DE-adducted DNA, Asp-192 forms a salt bridge with Arg-258.…”

Section: Pol ␤ Misinsertion Opposite the B[c]ph De-dgmentioning

confidence: 99%

Structure of DNA polymerase β with a benzo[ c ]phenanthrene diol epoxide-adducted template exhibits mutagenic features

Batra

Shock

Prasad

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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We have determined the crystal structure of the human base excision repair enzyme DNA polymerase ␤ (Pol ␤) in complex with a 1-nt gapped DNA substrate containing a template N 2 -guanine adduct of the tumorigenic (؊)-benzo[c]phenanthrene 4R,3S-diol 2S,1R-epoxide in the gap. Nucleotide insertion opposite this adduct favors incorrect purine nucleotides over the correct dCMP and hence can be mutagenic. The structure reveals that the phenanthrene ring system is stacked with the base pair immediately 3 to the modified guanine, thereby occluding the normal binding site for the correct incoming nucleoside triphosphate. The modified guanine base is displaced downstream and prevents the polymerase from achieving the catalytically competent closed conformation. The incoming nucleotide binding pocket is distorted, and the adducted deoxyguanosine is in a syn conformation, exposing its Hoogsteen edge, which can hydrogen-bond with dATP or dGTP. In a reconstituted base excision repair system, repair of a deaminated cytosine (i.e., uracil) opposite the adducted guanine was dramatically decreased at the Pol ␤ insertion step, but not blocked. The efficiency of gap-filling dCMP insertion opposite the adduct was diminished by >6 orders of magnitude compared with an unadducted templating guanine. In contrast, significant misinsertion of purine nucleotides (but not dTMP) opposite the adducted guanine was observed. Pol ␤ also misinserts a purine nucleotide opposite the adduct with ungapped DNA and exhibits limited bypass DNA synthesis. These results indicate that Pol ␤-dependent base excision repair of uracil opposite, or replication through, this bulky DNA adduct can be mutagenic.DNA adduct ͉ DNA repair ͉ fidelity ͉ mutagenesis P olycyclic aromatic hydrocarbons (PAHs) such as benzo- , and the adduct derived from trans-opening of the epoxide ring by the exocyclic 2-amino group of guanine in DNA, which is the subject of the present investigation.Bulky PAH-derived lesions can persist in human tissues when cellular repair enzymes fail to correct these lesions (13). The presence of these lesions in the genome blocks replicative DNA polymerases. If these lesions are not repaired, they must be bypassed for replication to continue. Bypass DNA synthesis occurs when a translesional DNA polymerase is recruited to the site of DNA damage and inserts a nucleotide opposite the adduct. DNA synthesis can be error-free or error-prone. The resulting base pair is then extended by either the same DNA polymerase or an auxiliary polymerase. Because replication of these adducts is known to result in mutations (14-16), mutagenic bypass provides a mechanism for adduct-induced tumor initiation and progression.Several DNA repair mechanisms protect cells from the deleterious effects of DNA lesions. For example, bulky DNA adducts such as PAH adducts and the formamidopyrimidine adduct of aflatoxin are primarily repaired by nucleotide excision repair (17, 18), although some unstable B[a]P DE adducts might promote depurination and elicit the base excision repair (BER...

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“…A new high-resolution structure (PDB accession code 2FMQ 9 ), in which Mg(c) is replaced by a Na + [Na(c)] ( Figure 1B), exhibits the same coordination geometry, suggesting that in the 1BPY structure the metal ion in the catalytic site was Na + rather than Mg 2+. 4,9 Computer simulations can help elucidate certain aspects of the function of DNA polymerases such as the effect of mutations on conformational changes 11,12 or energetics. [13][14][15][16] Magnesium ions are usually represented as ions with a formal point charge of +2 that interact with the protein environment and the substrate through nonbonded interactions.…”

Section: Introductionmentioning

confidence: 99%

Magnesium-cationic Dummy Atom Molecules Enhance Representation of DNA Polymerase β in Molecular Dynamics Simulations: Improved Accuracy in Studies of Structural Features and Mutational Effects

Oelschlaeger

Klähn

Beard

et al. 2007

Journal of Molecular Biology

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146

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SummaryHuman DNA polymerase β (pol β) fills gaps in DNA as part of base excision DNA repair. Due to its small size it is a convenient model enzyme for other DNA polymerases. Its active site contains two Mg 2+ ions, of which one binds an incoming dNTP and one catalyzes its condensation with the DNA primer strand. Simulating such binuclear metalloenzymes accurately but computationally efficiently is a challenging task. Here, we present a magnesium-cationic dummy atom approach that can easily be implemented in molecular mechanical force fields such as the ENZYMIX or the AMBER force fields. All properties investigated in this paper, that is, structure and energetics of both Michaelis complexes and transition state (TS) complexes were represented more accurately using the magnesium-cationic dummy atom model than using the traditional one-atom representation for Mg 2+ ions. The improved agreement between calculated free energies of binding of TS models to different pol β variants and the experimentally determined activation free energies indicates that this model will be useful in studying mutational effects on catalytic efficiency and fidelity of DNA polymerases. The model should also have broad applicability to the modeling of other magnesiumcontaining proteins.

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Magnesium-Induced Assembly of a Complete DNA Polymerase Catalytic Complex

Cited by 243 publications

References 48 publications

Role of the catalytic metal during polymerization by DNA polymerase lambda

Role of the catalytic metal during polymerization by DNA polymerase lambda

Structure of DNA polymerase β with a benzo[ c ]phenanthrene diol epoxide-adducted template exhibits mutagenic features

Magnesium-cationic Dummy Atom Molecules Enhance Representation of DNA Polymerase β in Molecular Dynamics Simulations: Improved Accuracy in Studies of Structural Features and Mutational Effects

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