Identification of a New Motif Required for the 3′–5′ Exonuclease Activity of Escherichia coli DNA Polymerase I (Klenow Fragment)

Kukreti, Pinky; Singh, Kamalendra; Ketkar, Amit; Modak, Mukund J.

doi:10.1074/jbc.m801053200

Cited by 17 publications

(12 citation statements)

References 28 publications

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“…This implies that the backtracking long pause is usually associated with the proofreading, which is consistent with the experimental data 10, 29, 34. This proofreading associated with the backtracking pause for RNAP corresponds to that associated with the 3′–5′ exonuclease activity for high‐fidelity DNA polymerase (DNAP),53, 54 and the fidelity resulting from the nucleotide insertion checkpoint55 is not considered here.…”

Section: Modelsupporting

confidence: 78%

Curing of mixtures of epoxy resins and 4‐methyl‐1,3‐dioxolan‐2‐one with several initiators

Cervellera

Ramis

Salla

et al. 2006

J of Applied Polymer Sci

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Diglycidyl ether of bisphenol A or 3,4-epoxycyclohexylmethyl 3,4-epoxycyclohexane carboxylate were mixed with different proportions of 4-methyl-1,3-dioxolan-2-one and cured using lanthanide triflates as initiators. In order to compare the materials obtained, conventional initiators such as boron trifluoride complexes and N,N-dimethylaminopyridine were also tested. The curing process was followed by differential scanning calorimetry (DSC) and Fourier transform IR in attenuated total reflectance mode. This technique proved that the carbonate accelerates the curing process because it helps to form the active initiating species, although it was not chemically incorporated into the network and remained entrapped in the material. The DSC kinetic study was also reported.

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Section: Modelsupporting

confidence: 78%

Curing of mixtures of epoxy resins and 4‐methyl‐1,3‐dioxolan‐2‐one with several initiators

Cervellera

Ramis

Salla

et al. 2006

J of Applied Polymer Sci

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“…In contrast the binding of ssDNA to Klenow was relatively weak, with an apparent K d of about 40 nM. This is very close to previously reported estimates of K d for ssDNA binding to Klenow in similar buffer conditions (36). We conclude that a significant component of the binding free energy of P/T junction DNA to Klenow is contributed by interactions involving the double-stranded portion of the construct.…”

Section: B-d)supporting

confidence: 70%

“…The protein residues interacting with the DNA in these sites are clearly different for the pol and exo mode-bound complexes (36,40). In this study of polymerase-DNA complexes in solution, we have characterized the local conformations of bases interacting with the first three polymerase binding sites listed above.…”

Section: Discussionmentioning

confidence: 99%

Local Conformations and Competitive Binding Affinities of Single- and Double-stranded Primer-Template DNA at the Polymerization and Editing Active Sites of DNA Polymerases

Datta

Johnson

LiCata

et al. 2009

Journal of Biological Chemistry

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In addition to their capacity for template-directed 5 3 3 DNA synthesis at the polymerase (pol) site, DNA polymerases have a separate 3 3 5 exonuclease (exo) editing activity that is involved in assuring the fidelity of DNA replication. Upon misincorporation of an incorrect nucleotide residue, the 3 terminus of the primer strand at the primer-template (P/T) junction is preferentially transferred to the exo site, where the faulty residue is excised, allowing the shortened primer to rebind to the template strand at the pol site and incorporate the correct dNTP. Here we describe the conformational changes that occur in the primer strand as it shuttles between the pol and exo sites of replication-competent Klenow and Klentaq DNA polymerase complexes in solution and use these conformational changes to measure the equilibrium distribution of the primer between these sites for P/T DNA constructs carrying both matched and mismatched primer termini. To this end, we have measured the fluorescence and circular dichroism spectra at wavelengths of >300 nm for conformational probes comprising pairs of 2-aminopurine bases site-specifically replacing adenine bases at various positions in the primer strand of P/T DNA constructs bound to DNA polymerases. Control experiments that compare primer conformations with available x-ray structures confirm the validity of this approach. These distributions and the conformational changes in the P/T DNA that occur during template-directed DNA synthesis in solution illuminate some of the mechanisms used by DNA polymerases to assure the fidelity of DNA synthesis. Escherichia coli DNA polymerase (DNAP)2 I is a single subunit polymerase that is organized into three functional domains: an N-terminal domain that is associated with 5Ј 3 3Ј exonuclease activity, an intermediate domain that carries the 3Ј 3 5Ј proofreading activity, and a C-terminal domain that is associated with the 5Ј 3 3Ј template-directed polymerization activity. An important role of DNAP I is to remove the RNA primers of the Okazaki fragments formed during lagging strand DNA synthesis in E. coli replication and to fill in the resulting gaps by template-directed DNA synthesis (1). An N-terminal deletion mutant of DNAP I, known as the "large fragment" or Klenow form of the enzyme, contains only the polymerase (pol) and the 3Ј 3 5Ј exonuclease (exo) domains. The Klenow polymerase has served and continues to serve as an excellent model system for isolating and defining general structure-function relationships in polymerases and in the supporting machinery of DNA replication.The main function of the 3Ј 3 5Ј exonuclease activity of DNAP I is to remove misincorporated nucleotide residues from the 3Ј-end of the primer (2), thus contributing significantly to the overall fidelity of DNA replication (3). Contrary to initial expectations, crystallographic studies showed that the pol and exo active sites are quite far apart in replication polymerases, about 30 Å in Klenow (4). As a consequence, the ability of polymerases to "shuttle" the 3Ј-...

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“…These exonucleases contain a basic loop motif adjacent to the active site that may facilitate melting of duplex DNA in order to provide a suitable single-stranded 3′ end for the active site [6] , [9] , [19] . Certainly, mutation of this basic loop in KF and Trex2 reduces exonuclease activity [9] , [21] , suggesting a role for the motif in binding nucleic acid, particularly the non-substrate strand as it extends from the active site [9] , [21] . We note that in Lassa NP, the site that is positionally equivalent to the basic loop of the other exonucleases is neither basic nor loop-shaped, but is instead much shorter and mostly hydrophobic ( Figure 7 ).…”

Section: Discussionmentioning

confidence: 99%

Structural Basis for the dsRNA Specificity of the Lassa Virus NP Exonuclease

et al. 2012

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Lassa virus causes hemorrhagic fever characterized by immunosuppression. The nucleoprotein of Lassa virus, termed NP, binds the viral genome. It also has an additional enzymatic activity as an exonuclease that specifically digests double-stranded RNA (dsRNA). dsRNA is a strong signal to the innate immune system of viral infection. Digestion of dsRNA by the NP exonuclease activity appears to cause suppression of innate immune signaling in the infected cell. Although the fold of the NP enzyme is conserved and the active site completely conserved with other exonucleases in its DEDDh family, NP is atypical among exonucleases in its preference for dsRNA and its strict specificity for one substrate. Here, we present the crystal structure of Lassa virus NP in complex with dsRNA. We find that unlike the exonuclease in Klenow fragment, the double-stranded nucleic acid in complex with Lassa NP remains base-paired instead of splitting, and that binding of the paired complementary strand is achieved by “relocation” of a basic loop motif from its typical exonuclease position. Further, we find that just one single glycine that contacts the substrate strand and one single tyrosine that stacks with a base of the complementary, non-substrate strand are responsible for the unique substrate specificity. This work thus provides templates for development of antiviral drugs that would be specific for viral, rather than host exonucleases of similar fold and active site, and illustrates how a very few amino acid changes confer alternate specificity and biological phenotype to an enzyme.

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Identification of a New Motif Required for the 3′–5′ Exonuclease Activity of Escherichia coli DNA Polymerase I (Klenow Fragment)

Cited by 17 publications

References 28 publications

Curing of mixtures of epoxy resins and 4‐methyl‐1,3‐dioxolan‐2‐one with several initiators

Curing of mixtures of epoxy resins and 4‐methyl‐1,3‐dioxolan‐2‐one with several initiators

Local Conformations and Competitive Binding Affinities of Single- and Double-stranded Primer-Template DNA at the Polymerization and Editing Active Sites of DNA Polymerases

Structural Basis for the dsRNA Specificity of the Lassa Virus NP Exonuclease

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