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Robert D Kuchta - One of the best experts on this subject based on the ideXlab platform.
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Polymerase and Exonuclease Activities in Herpes Simplex Virus Type 1 DNA Polymerase Are Not Highly Coordinated
2016Co-Authors: Ashwani Kumar Vashishtha, Robert D KuchtaAbstract:ABSTRACT: The herpes polymerase−processivity factor com-plex consists of the catalytic UL30 subunit containing both polymerase and proofreading Exonuclease activities and the UL42 subunit that acts as a processivity factor. Curiously, the highly active Exonuclease has minimal impact on the accumulation of mismatches generated by the polymerase activity. We utilized a series of oligonucleotides of defined sequence to define the interactions between the polymerase and Exonuclease active sites. Exonuclease activity requires unwinding of two nucleotides of the duplex primer−template. Surprisingly, even though the exonu-clease rate is much higher than the rate of DNA dissociation, the Exonuclease degrades both single- and double-stranded DNA in a nonprocessive manner. Efficient proofreading of incorrect nucleotides incorporated by the polymerase would seem to require efficient translocation of DNA between the Exonuclease and polymerase active sites. However, we found that translocation of DNA from the Exonuclease to polymerase active site i
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effects of acyclovir foscarnet and ribonucleotides on herpes simplex virus 1 dna polymerase mechanistic insights and a novel mechanism for preventing stable incorporation of ribonucleotides into dna
Biochemistry, 2016Co-Authors: Ashwani Kumar Vashishtha, Robert D KuchtaAbstract:We examined the impact of two clinically approved anti-herpes drugs, acyclovir and Forscarnet (phosphonoformate), on the Exonuclease activity of the herpes simplex virus-1 DNA polymerase, UL30. Acyclovir triphosphate and Foscarnet, along with the closely related phosphonoacetic acid, did not affect Exonuclease activity on single-stranded DNA. Furthermore, blocking the polymerase active site due to either binding of Foscarnet or phosphonoacetic acid to the E-DNA complex or polymerization of acyclovir onto the DNA also had a minimal effect on Exonuclease activity. The inability of the Exonuclease to excise acyclovir from the primer 3'-terminus results from the altered sugar structure directly impeding phosphodiester bond hydrolysis as opposed to inhibiting binding, unwinding of the DNA by the Exonuclease, or transfer of the DNA from the polymerase to the Exonuclease. Removing the 3'-hydroxyl or the 2'-carbon from the nucleotide at the 3'-terminus of the primer strongly inhibited Exonuclease activity, although addition of a 2'-hydroxyl did not affect Exonuclease activity. The biological consequences of these results are twofold. First, the ability of acyclovir and Foscarnet to block dNTP polymerization without impacting Exonuclease activity raises the possibility that their effects on herpes replication may involve both direct inhibition of dNTP polymerization and Exonuclease-mediated destruction of herpes DNA. Second, the ability of the Exonuclease to rapidly remove a ribonucleotide at the primer 3'-terminus in combination with the polymerase not efficiently adding dNTPs onto this primer provides a novel mechanism by which the herpes replication machinery can prevent incorporation of ribonucleotides into newly synthesized DNA.
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Effects of Acyclovir, Foscarnet, and Ribonucleotides on Herpes Simplex Virus‑1 DNA Polymerase: Mechanistic Insights and a Novel Mechanism for Preventing Stable Incorporation of Ribonucleotides into DNA
2016Co-Authors: Ashwani Kumar Vashishtha, Robert D KuchtaAbstract:We examined the impact of two clinically approved anti-herpes drugs, acyclovir and Forscarnet (phosphonoformate), on the Exonuclease activity of the herpes simplex virus-1 DNA polymerase, UL30. Acyclovir triphosphate and Foscarnet, along with the closely related phosphonoacetic acid, did not affect Exonuclease activity on single-stranded DNA. Furthermore, blocking the polymerase active site due to either binding of Foscarnet or phosphonoacetic acid to the E–DNA complex or polymerization of acyclovir onto the DNA also had a minimal effect on Exonuclease activity. The inability of the Exonuclease to excise acyclovir from the primer 3′-terminus results from the altered sugar structure directly impeding phosphodiester bond hydrolysis as opposed to inhibiting binding, unwinding of the DNA by the Exonuclease, or transfer of the DNA from the polymerase to the Exonuclease. Removing the 3′-hydroxyl or the 2′-carbon from the nucleotide at the 3′-terminus of the primer strongly inhibited Exonuclease activity, although addition of a 2′-hydroxyl did not affect Exonuclease activity. The biological consequences of these results are twofold. First, the ability of acyclovir and Foscarnet to block dNTP polymerization without impacting Exonuclease activity raises the possibility that their effects on herpes replication may involve both direct inhibition of dNTP polymerization and Exonuclease-mediated destruction of herpes DNA. Second, the ability of the Exonuclease to rapidly remove a ribonucleotide at the primer 3′-terminus in combination with the polymerase not efficiently adding dNTPs onto this primer provides a novel mechanism by which the herpes replication machinery can prevent incorporation of ribonucleotides into newly synthesized DNA
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polymerase and Exonuclease activities in herpes simplex virus type 1 dna polymerase are not highly coordinated
Biochemistry, 2015Co-Authors: Ashwani Kumar Vashishtha, Robert D KuchtaAbstract:The herpes polymerase-processivity factor complex consists of the catalytic UL30 subunit containing both polymerase and proofreading Exonuclease activities and the UL42 subunit that acts as a processivity factor. Curiously, the highly active Exonuclease has minimal impact on the accumulation of mismatches generated by the polymerase activity. We utilized a series of oligonucleotides of defined sequence to define the interactions between the polymerase and Exonuclease active sites. Exonuclease activity requires unwinding of two nucleotides of the duplex primer-template. Surprisingly, even though the Exonuclease rate is much higher than the rate of DNA dissociation, the Exonuclease degrades both single- and double-stranded DNA in a nonprocessive manner. Efficient proofreading of incorrect nucleotides incorporated by the polymerase would seem to require efficient translocation of DNA between the Exonuclease and polymerase active sites. However, we found that translocation of DNA from the Exonuclease to polymerase active site is remarkably inefficient. Consistent with inefficient translocation, the DNA binding sites for the Exonuclease and polymerase active sites appear to be largely independent, such that the two activities appear noncoordinated. Finally, the presence or absence of UL42 did not impact the coordination of the polymerase and Exonuclease activities. In addition to providing fundamental insights into how the polymerase and Exonuclease function together, these activities provide a rationale for understanding why the Exonuclease minimally impacts accumulation of mismatches by the purified polymerase and raise the question of how these two activities function together in vivo.
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dna polymerase epsilon aphidicolin inhibition and the relationship between polymerase and Exonuclease activity
Biochemistry, 1993Co-Authors: Chung Hui Cheng, Robert D KuchtaAbstract:: Calf thymus DNA polymerase epsilon readily uses short, synthetic oligonucleotides as substrates for both polymerase and Exonuclease activity. These substrates were used to examine the mechanism of inhibition by aphidicolin. Aphidicolin competes with each of the four dNTPs for binding to a pol epsilon.DNA complex. Importantly, aphidicolin binds equally well regardless of the identity of the next template base to be replicated (Ki approximately 0.6 microM). Hydrolysis of synthetic templates of defined sequence by the 3'-->5' Exonuclease was examined. pol epsilon preferred to hydrolyze single-stranded DNA 3-fold better than double-stranded DNA (Vmax/KM), while under Vmax conditions single-stranded DNA was hydrolyzed 100-fold faster than double-stranded DNA. Aphidicolin did not inhibit Exonuclease activity on single-stranded DNA; however, activity on double-stranded DNA was partially inhibited. Formation of an E.[template.primer].aphidicolin ternary complex inhibits Exonuclease activity. However, even under conditions where the polymerase site is completely blocked by a template-primer, the Exonuclease retains significant activity.
Ashwani Kumar Vashishtha - One of the best experts on this subject based on the ideXlab platform.
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Polymerase and Exonuclease Activities in Herpes Simplex Virus Type 1 DNA Polymerase Are Not Highly Coordinated
2016Co-Authors: Ashwani Kumar Vashishtha, Robert D KuchtaAbstract:ABSTRACT: The herpes polymerase−processivity factor com-plex consists of the catalytic UL30 subunit containing both polymerase and proofreading Exonuclease activities and the UL42 subunit that acts as a processivity factor. Curiously, the highly active Exonuclease has minimal impact on the accumulation of mismatches generated by the polymerase activity. We utilized a series of oligonucleotides of defined sequence to define the interactions between the polymerase and Exonuclease active sites. Exonuclease activity requires unwinding of two nucleotides of the duplex primer−template. Surprisingly, even though the exonu-clease rate is much higher than the rate of DNA dissociation, the Exonuclease degrades both single- and double-stranded DNA in a nonprocessive manner. Efficient proofreading of incorrect nucleotides incorporated by the polymerase would seem to require efficient translocation of DNA between the Exonuclease and polymerase active sites. However, we found that translocation of DNA from the Exonuclease to polymerase active site i
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effects of acyclovir foscarnet and ribonucleotides on herpes simplex virus 1 dna polymerase mechanistic insights and a novel mechanism for preventing stable incorporation of ribonucleotides into dna
Biochemistry, 2016Co-Authors: Ashwani Kumar Vashishtha, Robert D KuchtaAbstract:We examined the impact of two clinically approved anti-herpes drugs, acyclovir and Forscarnet (phosphonoformate), on the Exonuclease activity of the herpes simplex virus-1 DNA polymerase, UL30. Acyclovir triphosphate and Foscarnet, along with the closely related phosphonoacetic acid, did not affect Exonuclease activity on single-stranded DNA. Furthermore, blocking the polymerase active site due to either binding of Foscarnet or phosphonoacetic acid to the E-DNA complex or polymerization of acyclovir onto the DNA also had a minimal effect on Exonuclease activity. The inability of the Exonuclease to excise acyclovir from the primer 3'-terminus results from the altered sugar structure directly impeding phosphodiester bond hydrolysis as opposed to inhibiting binding, unwinding of the DNA by the Exonuclease, or transfer of the DNA from the polymerase to the Exonuclease. Removing the 3'-hydroxyl or the 2'-carbon from the nucleotide at the 3'-terminus of the primer strongly inhibited Exonuclease activity, although addition of a 2'-hydroxyl did not affect Exonuclease activity. The biological consequences of these results are twofold. First, the ability of acyclovir and Foscarnet to block dNTP polymerization without impacting Exonuclease activity raises the possibility that their effects on herpes replication may involve both direct inhibition of dNTP polymerization and Exonuclease-mediated destruction of herpes DNA. Second, the ability of the Exonuclease to rapidly remove a ribonucleotide at the primer 3'-terminus in combination with the polymerase not efficiently adding dNTPs onto this primer provides a novel mechanism by which the herpes replication machinery can prevent incorporation of ribonucleotides into newly synthesized DNA.
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Effects of Acyclovir, Foscarnet, and Ribonucleotides on Herpes Simplex Virus‑1 DNA Polymerase: Mechanistic Insights and a Novel Mechanism for Preventing Stable Incorporation of Ribonucleotides into DNA
2016Co-Authors: Ashwani Kumar Vashishtha, Robert D KuchtaAbstract:We examined the impact of two clinically approved anti-herpes drugs, acyclovir and Forscarnet (phosphonoformate), on the Exonuclease activity of the herpes simplex virus-1 DNA polymerase, UL30. Acyclovir triphosphate and Foscarnet, along with the closely related phosphonoacetic acid, did not affect Exonuclease activity on single-stranded DNA. Furthermore, blocking the polymerase active site due to either binding of Foscarnet or phosphonoacetic acid to the E–DNA complex or polymerization of acyclovir onto the DNA also had a minimal effect on Exonuclease activity. The inability of the Exonuclease to excise acyclovir from the primer 3′-terminus results from the altered sugar structure directly impeding phosphodiester bond hydrolysis as opposed to inhibiting binding, unwinding of the DNA by the Exonuclease, or transfer of the DNA from the polymerase to the Exonuclease. Removing the 3′-hydroxyl or the 2′-carbon from the nucleotide at the 3′-terminus of the primer strongly inhibited Exonuclease activity, although addition of a 2′-hydroxyl did not affect Exonuclease activity. The biological consequences of these results are twofold. First, the ability of acyclovir and Foscarnet to block dNTP polymerization without impacting Exonuclease activity raises the possibility that their effects on herpes replication may involve both direct inhibition of dNTP polymerization and Exonuclease-mediated destruction of herpes DNA. Second, the ability of the Exonuclease to rapidly remove a ribonucleotide at the primer 3′-terminus in combination with the polymerase not efficiently adding dNTPs onto this primer provides a novel mechanism by which the herpes replication machinery can prevent incorporation of ribonucleotides into newly synthesized DNA
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polymerase and Exonuclease activities in herpes simplex virus type 1 dna polymerase are not highly coordinated
Biochemistry, 2015Co-Authors: Ashwani Kumar Vashishtha, Robert D KuchtaAbstract:The herpes polymerase-processivity factor complex consists of the catalytic UL30 subunit containing both polymerase and proofreading Exonuclease activities and the UL42 subunit that acts as a processivity factor. Curiously, the highly active Exonuclease has minimal impact on the accumulation of mismatches generated by the polymerase activity. We utilized a series of oligonucleotides of defined sequence to define the interactions between the polymerase and Exonuclease active sites. Exonuclease activity requires unwinding of two nucleotides of the duplex primer-template. Surprisingly, even though the Exonuclease rate is much higher than the rate of DNA dissociation, the Exonuclease degrades both single- and double-stranded DNA in a nonprocessive manner. Efficient proofreading of incorrect nucleotides incorporated by the polymerase would seem to require efficient translocation of DNA between the Exonuclease and polymerase active sites. However, we found that translocation of DNA from the Exonuclease to polymerase active site is remarkably inefficient. Consistent with inefficient translocation, the DNA binding sites for the Exonuclease and polymerase active sites appear to be largely independent, such that the two activities appear noncoordinated. Finally, the presence or absence of UL42 did not impact the coordination of the polymerase and Exonuclease activities. In addition to providing fundamental insights into how the polymerase and Exonuclease function together, these activities provide a rationale for understanding why the Exonuclease minimally impacts accumulation of mismatches by the purified polymerase and raise the question of how these two activities function together in vivo.
Charles W Knopf - One of the best experts on this subject based on the ideXlab platform.
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herpes simplex virus type 1 dna polymerase mutational analysis of the 3 5 Exonuclease domain
Journal of Biological Chemistry, 1996Co-Authors: Frank J P Kuhn, Charles W KnopfAbstract:Like true DNA replicases, herpes simplex virus type 1 DNA polymerase is equipped with a proofreading 3′-5′-Exonuclease. In order to assess the functional significance of conserved residues in the putative Exonuclease domain, we introduced point mutations as well as deletions within and near the conserved motifs' Exonuclease (Exo) I, II, and III of the DNA polymerase gene from a phosphonoacetic acid-resistant derivative of herpes simplex virus-1 strain ANG. We examined the catalytic activities of the partially purified enzymes after overexpression by recombinant baculovirus. Mutations of the motifs' Exo I (D368A, E370A) and Exo III (Y577F, D581A) yielded enzymes without detectable and severely impaired 3′-5′-Exonuclease activities, respectively. Except for the Exo I mutations, all other Exo mutations examined affected both Exonuclease and polymerization activities. Mutant enzymes D368A, E370A, Y557S, and D581A showed a significant ability to extend mispaired primer termini. Mutation Y557S resulted in a strong reduction of the 3′-5′-Exonuclease activity and in a polymerase activity that was hyperresistant to phosphonoacetic acid. The results of the mutational analysis provide evidence for a tight linkage of polymerase and 3′-5′-Exonuclease activity in the herpesviral enzyme.
Naeem Rashid - One of the best experts on this subject based on the ideXlab platform.
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characterization of tk1646 a highly thermostable 3 5 single strand specific Exonuclease from thermococcus kodakarensis
International Journal of Biological Macromolecules, 2019Co-Authors: Muhammad Sulaiman Saeed, Naeem RashidAbstract:Abstract Exonucleases catalyze the hydrolysis of terminal phosphodiester bond in nucleic acid. They play important role in maintaining the integrity of DNA in eukaryotes, prokaryotes and archaea. Limited studies have been done on archaeal Exonucleases. Here we report molecular cloning of TK1646, a putative Exonuclease from the hyperthermophilic archaeon Thermococcus kodakarensis, and expression of the gene in Escherichia coli. Recombinant TK1646, produced in soluble and active form, was purified to apparent homogeneity. Characterization of the recombinant enzyme indicated that it was single strand specific 3′–5′ Exonuclease which cleaved the substrate DNA after every two nucleotides. It exhibited highest activity at 85–100 °C and pH 9.0. Unique property of TK1646 was its thermostability as it maintained its activity even at 100 °C with a half-life of 180 min. Recombinant TK1646 followed Michaelis-Menten kinetics and exhibited apparent Km and Vmax values of 33 ± 4 μM and 812 ± 48 nmol/min/mg, respectively. To the best of our knowledge this is the most thermostable single strand specific 3′–5′ Exonuclease characterized to date.
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characterization of tk1646 a highly thermostable 3 5 single strand specific Exonuclease from thermococcus kodakarensis
International Journal of Biological Macromolecules, 2019Co-Authors: Muhammad Sulaiman Saeed, Naeem RashidAbstract:Abstract Exonucleases catalyze the hydrolysis of terminal phosphodiester bond in nucleic acid. They play important role in maintaining the integrity of DNA in eukaryotes, prokaryotes and archaea. Limited studies have been done on archaeal Exonucleases. Here we report molecular cloning of TK1646, a putative Exonuclease from the hyperthermophilic archaeon Thermococcus kodakarensis, and expression of the gene in Escherichia coli. Recombinant TK1646, produced in soluble and active form, was purified to apparent homogeneity. Characterization of the recombinant enzyme indicated that it was single strand specific 3′–5′ Exonuclease which cleaved the substrate DNA after every two nucleotides. It exhibited highest activity at 85–100 °C and pH 9.0. Unique property of TK1646 was its thermostability as it maintained its activity even at 100 °C with a half-life of 180 min. Recombinant TK1646 followed Michaelis-Menten kinetics and exhibited apparent Km and Vmax values of 33 ± 4 μM and 812 ± 48 nmol/min/mg, respectively. To the best of our knowledge this is the most thermostable single strand specific 3′–5′ Exonuclease characterized to date.
Mukund J. Modak - One of the best experts on this subject based on the ideXlab platform.
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identification of a new motif required for the 3 5 Exonuclease activity of escherichia coli dna polymerase i klenow fragment the rrry motif is necessary for the binding of single stranded dna substrate and the template strand of the mismatched duplex
Journal of Biological Chemistry, 2008Co-Authors: Pinky Kukreti, Kamalendra Singh, Amit Ketkar, Mukund J. ModakAbstract:The Klenow fragment of Escherichia coli DNA polymerase I houses catalytic centers for both polymerase and 3'-5' Exonuclease activities that are separated by about 35 A. Upon the incorporation of a mismatched nucleotide, the primer terminus is transferred from the polymerase site to an Exonuclease site designed for excision of the mismatched nucleotides. The structural comparison of the binary complexes of DNA polymerases in the polymerase and the Exonuclease modes, together with a molecular modeling of the template strand overhang in Klenow fragment, indicated its binding in the region spanning residues 821-824. Since these residues are conserved in the "A" family DNA polymerases, we have designated this region as the RRRY motif. The alanine substitution of individual amino acid residues of this motif did not change the polymerase activity; however, the 3'-5' Exonuclease activity was reduced 2-29-fold, depending upon the site of mutation. The R821A and R822A/Y824A mutant enzymes showed maximum cleavage defect with single-stranded DNA, mainly due to a large decrease in the ssDNA binding affinity of these enzymes. Mismatch removal by these enzymes was only moderately affected. However, data from the Exonuclease-polymerase balance assays with mismatched template-primer suggest that the mutant enzymes are defective in switching mismatched primer from the polymerase to the Exonuclease site. Thus, the RRRY motif provides a binding track for substrate ssDNA and for nonsubstrate single-stranded template overhang, in a polarity-dependent manner. This binding then facilitates cleavage of the substrate at the Exonuclease site.
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contribution of polar residues of the j helix in the 3 5 Exonuclease activity of escherichia coli dna polymerase i klenow fragment q677 regulates the removal of terminal mismatch
Biochemistry, 2005Co-Authors: Kamalendra Singh, Mukund J. ModakAbstract:Previous structural and biochemical data indicate a participation of the J-helix of Escherichia coli pol I in primer positioning at the polymerase and Exonuclease sites. The J-helix contains three polar residues: N675, Q677, and N678. Preliminary characterization of alanine substitutions of these residues showed that only Q677A DNA polymerase has substantially decreased polymerase and increased Exonuclease activity. The Q677A enzyme had approximately 2- and approximately 5-fold greater Exonuclease activity than the wild type (WT) with mismatched and matched template-primers (TPs), respectively. N675A and N678A DNA polymerases did not differ significantly from the WT in these activities, despite the fact that both residues are seen to interact with the TP in various pol I-DNA complexes. Pre-steady-state kinetic measurements for the Exonuclease activity of WT and mutant enzymes indicated nearly identical DNA binding affinity for ssDNA and mismatched TPs. However, with a matched TP, Q677A DNA polymerase exhibited increased Exonuclease site affinity. The most important characteristic of Q677A DNA polymerase was its ability to continue cleavage into the matched region of the TP after mismatch excision, in contrast to the WT and other mutant enzymes. The increase in the Exonuclease activity of Q677A DNA polymerase was further determined not to be solely due to the weakened binding at the polymerase site, by comparison with another polymerase-defective mutant enzyme, namely, R668A DNA polymerase. These enzymes have significantly decreased DNA binding affinity at the polymerase site, yet the Exonuclease activity parameters of R668A DNA polymerase remain similar to those of the WT. These results strongly suggest that participation of Q677 is required for positioning the primer terminus (a) in the polymerase site for continued nucleotide addition and (b) in the 3'-Exonuclease site for the controlled removal of mismatched nucleotides.