Kinetic Characterization of the Polymerase and Exonuclease Activities of the Gene 43 Protein of Bacteriophage T4

Todd L. Capson, James A. Peliska, Barbara Fenn Kaboord, Michelle West Frey, Chris Lively, Michael Dahlberg, Stephen J. Benkovic

Research output: Contribution to journalArticle

214 Citations (Scopus)

Abstract

The DNA polymerase from the bacteriophage T4 is part of a multienzyme complex required for the synthesis of DNA. As a first step in understanding the contributions of individual proteins to the dynamic properties of the complex, e.g., turnover, processivity, and fidelity of replication, the minimal kinetic schemes for the polymerase and exonuclease activities of the gene 43 protein have been determined by pre-steady-state kinetic methods and fit by computer simulation. A DNA primer/template (13/20-mer) was used as substrate; duplexes that contained more single-strand DNA resulted in nonproductive binding of the polymerase. The reaction sequence features an ordered addition of 13/20-mer followed by dATP to the T4 enzyme (dissociation constants of 70 nM and 20 μM) followed by rapid conversion (400 s−1) of the T4-13/20-mer-dATP complex to the T4-14/20-mer-PPiproduct species. A slow step (2 s−1) following PPirelease limits a single turnover, although this step is bypassed in multiple incorporations (13/20-mer → 17/20-mer) which occur at rates 400 s−1. Competition between correct versus incorrect nucleotides relative to the template strand indicates that the dissociation constants for the incorrect nucleotides are at millimolar values, thus providing evidence that the T4 polymerase, like the T7 but unlike the Klenow fragment polymerases, discriminates by factors 103against misincorporation in the nucleotide binding step. The exonuclease activity of the T4 enzyme requires an activation step, i.e., T4-DNA → T4-(DNA)*, whose rate constants reflect whether the 3ʹ-terminus of the primer is matched or mismatched; for matched 13/20-mer the constant is 1 s_1, and for mismatched 13T/20-mer, 5s−1. Evidence is presented from crossover experiments that this step may represent a melting of the terminus of the duplex, which is followed by rapid exonucleolytic cleavage (100 s−1). In the presence of the correct dNTP, primer extension is the rate-limiting step rather than a step involving travel of the duplex between separated exonuclease and polymerase sites. Since the rate constant for 13/20-mer or 13T/20-mer dissociation from the enzyme is 6 or 8 s−1 and competes with that for activation, the exonucleolytic editing by the enzyme alone in a single pass is somewhat inefficient (5 s−1/(8 s−1+ 5 s−1)), ca. 40%. Consequently, a major role for the accessory proteins may be to slow the rate of enzyme-substrate dissociation, thereby increasing overall fidelity and processivity.

Original languageEnglish (US)
Pages (from-to)10984-10994
Number of pages11
JournalBiochemistry
Volume31
Issue number45
DOIs
StatePublished - Feb 1 1992

Fingerprint

Exonucleases
Kinetics
Nucleotides
DNA
Enzymes
Multienzyme Complexes
Rate constants
DNA Polymerase I
Bacteriophage T4
Proteins
Chemical activation
Enzyme Activation
DNA Primers
DNA-Directed DNA Polymerase
Bacteriophages
Computer Simulation
Freezing
Accessories
Substrates
Melting

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Capson, T. L., Peliska, J. A., Kaboord, B. F., Frey, M. W., Lively, C., Dahlberg, M., & Benkovic, S. J. (1992). Kinetic Characterization of the Polymerase and Exonuclease Activities of the Gene 43 Protein of Bacteriophage T4. Biochemistry, 31(45), 10984-10994. https://doi.org/10.1021/bi00160a007
Capson, Todd L. ; Peliska, James A. ; Kaboord, Barbara Fenn ; Frey, Michelle West ; Lively, Chris ; Dahlberg, Michael ; Benkovic, Stephen J. / Kinetic Characterization of the Polymerase and Exonuclease Activities of the Gene 43 Protein of Bacteriophage T4. In: Biochemistry. 1992 ; Vol. 31, No. 45. pp. 10984-10994.
@article{e570c4d6bfe44c58b02567b327a980fa,
title = "Kinetic Characterization of the Polymerase and Exonuclease Activities of the Gene 43 Protein of Bacteriophage T4",
abstract = "The DNA polymerase from the bacteriophage T4 is part of a multienzyme complex required for the synthesis of DNA. As a first step in understanding the contributions of individual proteins to the dynamic properties of the complex, e.g., turnover, processivity, and fidelity of replication, the minimal kinetic schemes for the polymerase and exonuclease activities of the gene 43 protein have been determined by pre-steady-state kinetic methods and fit by computer simulation. A DNA primer/template (13/20-mer) was used as substrate; duplexes that contained more single-strand DNA resulted in nonproductive binding of the polymerase. The reaction sequence features an ordered addition of 13/20-mer followed by dATP to the T4 enzyme (dissociation constants of 70 nM and 20 μM) followed by rapid conversion (400 s−1) of the T4-13/20-mer-dATP complex to the T4-14/20-mer-PPiproduct species. A slow step (2 s−1) following PPirelease limits a single turnover, although this step is bypassed in multiple incorporations (13/20-mer → 17/20-mer) which occur at rates 400 s−1. Competition between correct versus incorrect nucleotides relative to the template strand indicates that the dissociation constants for the incorrect nucleotides are at millimolar values, thus providing evidence that the T4 polymerase, like the T7 but unlike the Klenow fragment polymerases, discriminates by factors 103against misincorporation in the nucleotide binding step. The exonuclease activity of the T4 enzyme requires an activation step, i.e., T4-DNA → T4-(DNA)*, whose rate constants reflect whether the 3ʹ-terminus of the primer is matched or mismatched; for matched 13/20-mer the constant is 1 s_1, and for mismatched 13T/20-mer, 5s−1. Evidence is presented from crossover experiments that this step may represent a melting of the terminus of the duplex, which is followed by rapid exonucleolytic cleavage (100 s−1). In the presence of the correct dNTP, primer extension is the rate-limiting step rather than a step involving travel of the duplex between separated exonuclease and polymerase sites. Since the rate constant for 13/20-mer or 13T/20-mer dissociation from the enzyme is 6 or 8 s−1 and competes with that for activation, the exonucleolytic editing by the enzyme alone in a single pass is somewhat inefficient (5 s−1/(8 s−1+ 5 s−1)), ca. 40{\%}. Consequently, a major role for the accessory proteins may be to slow the rate of enzyme-substrate dissociation, thereby increasing overall fidelity and processivity.",
author = "Capson, {Todd L.} and Peliska, {James A.} and Kaboord, {Barbara Fenn} and Frey, {Michelle West} and Chris Lively and Michael Dahlberg and Benkovic, {Stephen J.}",
year = "1992",
month = "2",
day = "1",
doi = "10.1021/bi00160a007",
language = "English (US)",
volume = "31",
pages = "10984--10994",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "45",

}

Capson, TL, Peliska, JA, Kaboord, BF, Frey, MW, Lively, C, Dahlberg, M & Benkovic, SJ 1992, 'Kinetic Characterization of the Polymerase and Exonuclease Activities of the Gene 43 Protein of Bacteriophage T4', Biochemistry, vol. 31, no. 45, pp. 10984-10994. https://doi.org/10.1021/bi00160a007

Kinetic Characterization of the Polymerase and Exonuclease Activities of the Gene 43 Protein of Bacteriophage T4. / Capson, Todd L.; Peliska, James A.; Kaboord, Barbara Fenn; Frey, Michelle West; Lively, Chris; Dahlberg, Michael; Benkovic, Stephen J.

In: Biochemistry, Vol. 31, No. 45, 01.02.1992, p. 10984-10994.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Kinetic Characterization of the Polymerase and Exonuclease Activities of the Gene 43 Protein of Bacteriophage T4

AU - Capson, Todd L.

AU - Peliska, James A.

AU - Kaboord, Barbara Fenn

AU - Frey, Michelle West

AU - Lively, Chris

AU - Dahlberg, Michael

AU - Benkovic, Stephen J.

PY - 1992/2/1

Y1 - 1992/2/1

N2 - The DNA polymerase from the bacteriophage T4 is part of a multienzyme complex required for the synthesis of DNA. As a first step in understanding the contributions of individual proteins to the dynamic properties of the complex, e.g., turnover, processivity, and fidelity of replication, the minimal kinetic schemes for the polymerase and exonuclease activities of the gene 43 protein have been determined by pre-steady-state kinetic methods and fit by computer simulation. A DNA primer/template (13/20-mer) was used as substrate; duplexes that contained more single-strand DNA resulted in nonproductive binding of the polymerase. The reaction sequence features an ordered addition of 13/20-mer followed by dATP to the T4 enzyme (dissociation constants of 70 nM and 20 μM) followed by rapid conversion (400 s−1) of the T4-13/20-mer-dATP complex to the T4-14/20-mer-PPiproduct species. A slow step (2 s−1) following PPirelease limits a single turnover, although this step is bypassed in multiple incorporations (13/20-mer → 17/20-mer) which occur at rates 400 s−1. Competition between correct versus incorrect nucleotides relative to the template strand indicates that the dissociation constants for the incorrect nucleotides are at millimolar values, thus providing evidence that the T4 polymerase, like the T7 but unlike the Klenow fragment polymerases, discriminates by factors 103against misincorporation in the nucleotide binding step. The exonuclease activity of the T4 enzyme requires an activation step, i.e., T4-DNA → T4-(DNA)*, whose rate constants reflect whether the 3ʹ-terminus of the primer is matched or mismatched; for matched 13/20-mer the constant is 1 s_1, and for mismatched 13T/20-mer, 5s−1. Evidence is presented from crossover experiments that this step may represent a melting of the terminus of the duplex, which is followed by rapid exonucleolytic cleavage (100 s−1). In the presence of the correct dNTP, primer extension is the rate-limiting step rather than a step involving travel of the duplex between separated exonuclease and polymerase sites. Since the rate constant for 13/20-mer or 13T/20-mer dissociation from the enzyme is 6 or 8 s−1 and competes with that for activation, the exonucleolytic editing by the enzyme alone in a single pass is somewhat inefficient (5 s−1/(8 s−1+ 5 s−1)), ca. 40%. Consequently, a major role for the accessory proteins may be to slow the rate of enzyme-substrate dissociation, thereby increasing overall fidelity and processivity.

AB - The DNA polymerase from the bacteriophage T4 is part of a multienzyme complex required for the synthesis of DNA. As a first step in understanding the contributions of individual proteins to the dynamic properties of the complex, e.g., turnover, processivity, and fidelity of replication, the minimal kinetic schemes for the polymerase and exonuclease activities of the gene 43 protein have been determined by pre-steady-state kinetic methods and fit by computer simulation. A DNA primer/template (13/20-mer) was used as substrate; duplexes that contained more single-strand DNA resulted in nonproductive binding of the polymerase. The reaction sequence features an ordered addition of 13/20-mer followed by dATP to the T4 enzyme (dissociation constants of 70 nM and 20 μM) followed by rapid conversion (400 s−1) of the T4-13/20-mer-dATP complex to the T4-14/20-mer-PPiproduct species. A slow step (2 s−1) following PPirelease limits a single turnover, although this step is bypassed in multiple incorporations (13/20-mer → 17/20-mer) which occur at rates 400 s−1. Competition between correct versus incorrect nucleotides relative to the template strand indicates that the dissociation constants for the incorrect nucleotides are at millimolar values, thus providing evidence that the T4 polymerase, like the T7 but unlike the Klenow fragment polymerases, discriminates by factors 103against misincorporation in the nucleotide binding step. The exonuclease activity of the T4 enzyme requires an activation step, i.e., T4-DNA → T4-(DNA)*, whose rate constants reflect whether the 3ʹ-terminus of the primer is matched or mismatched; for matched 13/20-mer the constant is 1 s_1, and for mismatched 13T/20-mer, 5s−1. Evidence is presented from crossover experiments that this step may represent a melting of the terminus of the duplex, which is followed by rapid exonucleolytic cleavage (100 s−1). In the presence of the correct dNTP, primer extension is the rate-limiting step rather than a step involving travel of the duplex between separated exonuclease and polymerase sites. Since the rate constant for 13/20-mer or 13T/20-mer dissociation from the enzyme is 6 or 8 s−1 and competes with that for activation, the exonucleolytic editing by the enzyme alone in a single pass is somewhat inefficient (5 s−1/(8 s−1+ 5 s−1)), ca. 40%. Consequently, a major role for the accessory proteins may be to slow the rate of enzyme-substrate dissociation, thereby increasing overall fidelity and processivity.

UR - http://www.scopus.com/inward/record.url?scp=0026464542&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026464542&partnerID=8YFLogxK

U2 - 10.1021/bi00160a007

DO - 10.1021/bi00160a007

M3 - Article

C2 - 1332748

AN - SCOPUS:0026464542

VL - 31

SP - 10984

EP - 10994

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 45

ER -

Capson TL, Peliska JA, Kaboord BF, Frey MW, Lively C, Dahlberg M et al. Kinetic Characterization of the Polymerase and Exonuclease Activities of the Gene 43 Protein of Bacteriophage T4. Biochemistry. 1992 Feb 1;31(45):10984-10994. https://doi.org/10.1021/bi00160a007