DNA Substrate Structural Requirements for the Exonuclease and Polymerase Activities of Procaryotic and Phage DNA Polymerases

Marlon Cowart, Katharine J. Gibson, Dwayne J. Allen, Stephen Benkovic

Research output: Contribution to journalArticle

93 Citations (Scopus)

Abstract

A DNA duplex covalently cross-linked between specific bases has been prepared. This and similar duplexes are substrates for the polymerase and exonuclease activities of the Klenow fragment of Escherichia coli DNA polymerase I and T4 and T7 DNA polymerases. The action of Klenow fragment on these duplexes indicates that the polymerase site does not require that the DNA duplex undergo strand separation for activity, whereas the exonuclease site requires that at least four base pairs of the primer strand must melt out for the exonucleolytic removal of nucleotides from the primer terminus. The exonucleolytic action of T4 and T7 DNA polymerases requires that only two and three bases respectively melt out for excision of nucleotides from the primer terminus. Klenow fragment and T4 DNA polymerase are able to polymerize onto duplexes incapaole of strand separation, whereas T7 DNA polymerase seems to require that the primer terminus be at least three bases from the cross-linked base pair. A DNA duplex with a biotin covalently linked to a specific base has been prepared. In the presence of the biotin binding protein avidin, the exonucleolytic activity of Klenow fragment requires that the primer terminus be at least 15 base pairs downstream from the base with the biotin-avidin complex. On the other hand, the polymerase activity of Klenow fragment required that the primer terminus be at least six base pairs downstream from the base with the biotin-avidin complex. These results suggest that the polymerase and exonuclease sites of Klenow are physically separate in solution and exhibit different substrate structural requirements for activity.

Original languageEnglish (US)
Pages (from-to)1975-1983
Number of pages9
JournalBiochemistry
Volume28
Issue number5
DOIs
StatePublished - Jan 1 1989

Fingerprint

DNA Polymerase I
Exonucleases
Bacteriophages
DNA-Directed DNA Polymerase
Base Pairing
Avidin
DNA
Substrates
Biotin
Nucleotides
Escherichia coli

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Cowart, Marlon ; Gibson, Katharine J. ; Allen, Dwayne J. ; Benkovic, Stephen. / DNA Substrate Structural Requirements for the Exonuclease and Polymerase Activities of Procaryotic and Phage DNA Polymerases. In: Biochemistry. 1989 ; Vol. 28, No. 5. pp. 1975-1983.
@article{7dc854e84baa44dd82ac228e7c09dcc5,
title = "DNA Substrate Structural Requirements for the Exonuclease and Polymerase Activities of Procaryotic and Phage DNA Polymerases",
abstract = "A DNA duplex covalently cross-linked between specific bases has been prepared. This and similar duplexes are substrates for the polymerase and exonuclease activities of the Klenow fragment of Escherichia coli DNA polymerase I and T4 and T7 DNA polymerases. The action of Klenow fragment on these duplexes indicates that the polymerase site does not require that the DNA duplex undergo strand separation for activity, whereas the exonuclease site requires that at least four base pairs of the primer strand must melt out for the exonucleolytic removal of nucleotides from the primer terminus. The exonucleolytic action of T4 and T7 DNA polymerases requires that only two and three bases respectively melt out for excision of nucleotides from the primer terminus. Klenow fragment and T4 DNA polymerase are able to polymerize onto duplexes incapaole of strand separation, whereas T7 DNA polymerase seems to require that the primer terminus be at least three bases from the cross-linked base pair. A DNA duplex with a biotin covalently linked to a specific base has been prepared. In the presence of the biotin binding protein avidin, the exonucleolytic activity of Klenow fragment requires that the primer terminus be at least 15 base pairs downstream from the base with the biotin-avidin complex. On the other hand, the polymerase activity of Klenow fragment required that the primer terminus be at least six base pairs downstream from the base with the biotin-avidin complex. These results suggest that the polymerase and exonuclease sites of Klenow are physically separate in solution and exhibit different substrate structural requirements for activity.",
author = "Marlon Cowart and Gibson, {Katharine J.} and Allen, {Dwayne J.} and Stephen Benkovic",
year = "1989",
month = "1",
day = "1",
doi = "10.1021/bi00431a004",
language = "English (US)",
volume = "28",
pages = "1975--1983",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "5",

}

DNA Substrate Structural Requirements for the Exonuclease and Polymerase Activities of Procaryotic and Phage DNA Polymerases. / Cowart, Marlon; Gibson, Katharine J.; Allen, Dwayne J.; Benkovic, Stephen.

In: Biochemistry, Vol. 28, No. 5, 01.01.1989, p. 1975-1983.

Research output: Contribution to journalArticle

TY - JOUR

T1 - DNA Substrate Structural Requirements for the Exonuclease and Polymerase Activities of Procaryotic and Phage DNA Polymerases

AU - Cowart, Marlon

AU - Gibson, Katharine J.

AU - Allen, Dwayne J.

AU - Benkovic, Stephen

PY - 1989/1/1

Y1 - 1989/1/1

N2 - A DNA duplex covalently cross-linked between specific bases has been prepared. This and similar duplexes are substrates for the polymerase and exonuclease activities of the Klenow fragment of Escherichia coli DNA polymerase I and T4 and T7 DNA polymerases. The action of Klenow fragment on these duplexes indicates that the polymerase site does not require that the DNA duplex undergo strand separation for activity, whereas the exonuclease site requires that at least four base pairs of the primer strand must melt out for the exonucleolytic removal of nucleotides from the primer terminus. The exonucleolytic action of T4 and T7 DNA polymerases requires that only two and three bases respectively melt out for excision of nucleotides from the primer terminus. Klenow fragment and T4 DNA polymerase are able to polymerize onto duplexes incapaole of strand separation, whereas T7 DNA polymerase seems to require that the primer terminus be at least three bases from the cross-linked base pair. A DNA duplex with a biotin covalently linked to a specific base has been prepared. In the presence of the biotin binding protein avidin, the exonucleolytic activity of Klenow fragment requires that the primer terminus be at least 15 base pairs downstream from the base with the biotin-avidin complex. On the other hand, the polymerase activity of Klenow fragment required that the primer terminus be at least six base pairs downstream from the base with the biotin-avidin complex. These results suggest that the polymerase and exonuclease sites of Klenow are physically separate in solution and exhibit different substrate structural requirements for activity.

AB - A DNA duplex covalently cross-linked between specific bases has been prepared. This and similar duplexes are substrates for the polymerase and exonuclease activities of the Klenow fragment of Escherichia coli DNA polymerase I and T4 and T7 DNA polymerases. The action of Klenow fragment on these duplexes indicates that the polymerase site does not require that the DNA duplex undergo strand separation for activity, whereas the exonuclease site requires that at least four base pairs of the primer strand must melt out for the exonucleolytic removal of nucleotides from the primer terminus. The exonucleolytic action of T4 and T7 DNA polymerases requires that only two and three bases respectively melt out for excision of nucleotides from the primer terminus. Klenow fragment and T4 DNA polymerase are able to polymerize onto duplexes incapaole of strand separation, whereas T7 DNA polymerase seems to require that the primer terminus be at least three bases from the cross-linked base pair. A DNA duplex with a biotin covalently linked to a specific base has been prepared. In the presence of the biotin binding protein avidin, the exonucleolytic activity of Klenow fragment requires that the primer terminus be at least 15 base pairs downstream from the base with the biotin-avidin complex. On the other hand, the polymerase activity of Klenow fragment required that the primer terminus be at least six base pairs downstream from the base with the biotin-avidin complex. These results suggest that the polymerase and exonuclease sites of Klenow are physically separate in solution and exhibit different substrate structural requirements for activity.

UR - http://www.scopus.com/inward/record.url?scp=0024549097&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024549097&partnerID=8YFLogxK

U2 - 10.1021/bi00431a004

DO - 10.1021/bi00431a004

M3 - Article

C2 - 2541768

AN - SCOPUS:0024549097

VL - 28

SP - 1975

EP - 1983

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 5

ER -