Structure of DNA Polymerase I Klenow Fragment Bound to Duplex DNA

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

doi:10.1126/science.8469987

Cited by 493 publications

(482 citation statements)

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“…4, B-E (solid lines), and show that the obtained rates fit our data very well. In conclusion, our kinetic model reveals new, to our knowledge, pathways in the mechanochemistry of T7 DNAp, and is consistent with our single-molecule data as well as previous biochemical (6,15) and structural (20,21) studies.…”

Section: Kinetic Model Of T7 Dnap For Replication Proofreading and supporting

confidence: 91%

“…The DNA length changes are all in the subnanometer range. For example, the DNA length change that occurs between pol and exo activities is consistent with that of the melting of~3 bp (0.62 nm), in agreement with structural data (20,21). Moreover, we can use the obtained rates to make predictions about the probability of binding in exo (Fig.…”

Section: Kinetic Model Of T7 Dnap For Replication Proofreading and supporting

confidence: 84%

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Switching between Exonucleolysis and Replication by T7 DNA Polymerase Ensures High Fidelity

Hoekstra

Depken

Lin

et al. 2017

Biophysical Journal

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DNA polymerase catalyzes the accurate transfer of genetic information from one generation to the next, and thus it is vitally important for replication to be faithful. DNA polymerase fulfills the strict requirements for fidelity by a combination of mechanisms: 1) high selectivity for correct nucleotide incorporation, 2) a slowing down of the replication rate after misincorporation, and 3) proofreading by excision of misincorporated bases. To elucidate the kinetic interplay between replication and proofreading, we used high-resolution optical tweezers to probe how DNA-duplex stability affects replication by bacteriophage T7 DNA polymerase. Our data show highly irregular replication dynamics, with frequent pauses and direction reversals as the polymerase cycles through the states that govern the mechanochemistry behind high-fidelity T7 DNA replication. We constructed a kinetic model that incorporates both existing biochemical data and the, to our knowledge, novel states we observed. We fit the model directly to the acquired pause-time and run-time distributions. Our findings indicate that the main pathway for error correction is DNA polymerase dissociation-mediated DNA transfer, followed by biased binding into the exonuclease active site. The number of bases removed by this proofreading mechanism is much larger than the number of erroneous bases that would be expected to be incorporated, ensuring a high-fidelity replication of the bacteriophage T7 genome.

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Section: Kinetic Model Of T7 Dnap For Replication Proofreading and supporting

confidence: 91%

Section: Kinetic Model Of T7 Dnap For Replication Proofreading and supporting

confidence: 84%

Switching between Exonucleolysis and Replication by T7 DNA Polymerase Ensures High Fidelity

Hoekstra

Depken

Lin

et al. 2017

Biophysical Journal

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“…Such interactions are common to nucleotide-binding proteins; anionic interaction with a nucleotide hydroxyl group, [30][31][32] and aromatic stacking against a nucleotide base occur in the DNA repair enzymes, 33,34 γ-tubulin, 35 the U1A spliceosomal protein, 36 the purine and pyrimidine phosphoribosyltransferases 32,37 and aspartyl-tRNA synthetase. 38 Indeed, the binding configuration at the SRP GTPase external nucleotide site is specifically reminiscent of similar arrangements in crystal structures of the editing complex of Klenow fragment 34,39 and AMP-bound adenine phosphoribosyltransferase (APRT) 40 ( Figure 6). In the 3′ to 5′ exonuclease active site of the Klenow fragment, the 3′-terminal base of the single-stranded DNA is positioned between Leu361 and Phe473, and the 2′-OH interacts with Glu357 ( Figure 6(b)).…”

Section: Gmp Is Bound On the Surface Of The Gtpase Heterodimer At A Cmentioning

confidence: 79%

Structure of a GDP:AlF4 Complex of the SRP GTPases Ffh and FtsY, and Identification of a Peripheral Nucleotide Interaction Site

Focia

Gawronski-Salerno

Coon

et al. 2006

Journal of Molecular Biology

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The signal recognition particle (SRP) GTPases Ffh and FtsY play a central role in co-translational targeting of proteins, assembling in a GTP-dependent manner to generate the SRP targeting complex at the membrane. A suite of residues in FtsY have been identified that are essential for the hydrolysis of GTP that accompanies disengagement. We have argued previously on structural grounds that this region mediates interactions that serve to activate the complex for disengagement and term it the activation region. We report here the structure of a complex of the SRP GTPases formed in the presence of GDP:AlF 4 . This complex accommodates the putative transition-state analog without undergoing significant change from the structure of the ground-state complex formed in the presence of the GTP analog GMPPCP. However, small shifts that do occur within the shared catalytic chamber may be functionally important. Remarkably, an external nucleotide interaction site was identified at the activation region, revealed by an unexpected contaminating GMP molecule bound adjacent to the catalytic chamber. This site exhibits conserved sequence and structural features that suggest a direct interaction with RNA plays a role in regulating the activity of the SRP targeting complex.

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“…Biochemical studies suggest that 5-8 base pairs of duplex DNA are covered by E. coli Klenow fragment when the primer terminus is in the pol active site (33). In the co-crystal structure of an editing complex, the 3Ј terminus is bound in the exo active site, whereas the duplex portion of the template-primer occupies the cleft between exo and pol domains similar to binding in the pol mode (18,40). Importantly, the extent of single-stranded DNA binding in the 3Ј-5Ј exo active site dictates that three or four base pairs of the duplex DNA must be melted before binding occurs (41,42).…”

Section: Discussionmentioning

confidence: 99%

Mutations in the Spacer Region of Drosophila Mitochondrial DNA Polymerase Affect DNA Binding, Processivity, and the Balance between Pol and Exo Function

Luo

Kaguni

2005

Journal of Biological Chemistry

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The catalytic subunit (␣) of mitochondrial DNA polymerase (pol ␥) shares conserved DNA polymerase and 3 -5 exonuclease active site motifs with Escherichia coli DNA polymerase I and bacteriophage T7 DNA polymerase. A major difference between the prokaryotic and mitochondrial proteins is the size and sequence of the region between the exonuclease and DNA polymerase domains, referred to as the spacer in pol ␥-␣. Four ␥-specific conserved sequence elements are located within the spacer region of the catalytic subunit in eukaryotic species from yeast to humans. To elucidate the functional roles of the spacer region, we pursued deletion and site-directed mutagenesis of Drosophila pol ␥. Mutant proteins were expressed from baculovirus constructs in insect cells, purified to near homogeneity, and analyzed biochemically. We find that mutations in three of the four conserved sequence elements within the spacer alter enzyme activity, processivity, and/or DNA binding affinity. In addition, several mutations affect differentially DNA polymerase and exonuclease activity and/or functional interactions with mitochondrial singlestranded DNA-binding protein. Based on these results and crystallographic evidence showing that the templateprimer binds in a cleft between the exonuclease and DNA polymerase domains in family A DNA polymerases, we propose that conserved sequences within the spacer of pol ␥ may position the substrate with respect to the enzyme catalytic domains.The mitochondrion is the eukaryotic organelle that carries out oxidative phosphorylation, fulfilling cellular requirements for energy production. Disruption of mitochondrial energy metabolism can occur by genetic or biochemical mechanisms and is associated with human disorders including degenerative diseases, cancer, and aging (1). Mutations in both the mitochondrial genome and in nuclear genes whose products have mitochondrial functions are linked to mitochondrial disease syndromes. Nuclear genes include those encoding the adenine nucleotide translocator 1 (2), thymidine phosphorylase (3), mitochondrial DNA helicase (Twinkle) (4), and the catalytic subunit of mitochondrial DNA polymerase (pol 1 ␥) (5). Pol ␥ has also been shown to be a target of oxidative damage, which reduces DNA polymerase activity in the mitochondrial matrix (6).Pol ␥ is the only DNA polymerase known to be required for mitochondrial DNA replication in animals (7). It is a member of the family A DNA polymerase group (8, 9), of which Escherichia coli pol I is the prototype (10). The crystal structures of numerous family A DNA polymerases have been solved, revealing a high degree of structural similarity among its members (11)(12)(13)(14)(15)(16)(17). The interaction between DNA polymerase and template-primer DNA has also been revealed in molecular detail by structural determination of pol:DNA co-crystals and in biochemical studies. The pol domain comprises palm, fingers, and thumb subdomains that are separated from the 3Ј-5Ј exonuclease (exo) domain by a cleft that binds the template-primer. Wher...

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Structure of DNA Polymerase I Klenow Fragment Bound to Duplex DNA

Cited by 493 publications

References 28 publications

Switching between Exonucleolysis and Replication by T7 DNA Polymerase Ensures High Fidelity

Switching between Exonucleolysis and Replication by T7 DNA Polymerase Ensures High Fidelity

Structure of a GDP:AlF4 Complex of the SRP GTPases Ffh and FtsY, and Identification of a Peripheral Nucleotide Interaction Site

Mutations in the Spacer Region of Drosophila Mitochondrial DNA Polymerase Affect DNA Binding, Processivity, and the Balance between Pol and Exo Function

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