Structure-specific DNA binding by bacteriophage T5 5'→3' exonuclease

Scott J. Garforth, Jon R. Sayers

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Phage T5 exonuclease is a 5'→3' exodeoxyribonuclease that also exhibits endonucleolytic activity on flap structures (branched duplex DNA containing a free single-stranded 5'-end). Oligonucleotides were used to construct duplexes with either blunt ends, 5'-overhangs, 3'-overhangs, a flap or a forked end (pseudo-Y). The binding of T5 exonuclease to various structures was investigated using native electrophoretic mobility shift assays (EMSA) in the absence of the essential divalent metal cofactor. Binding of T5 exonuclease to either blunt-ended duplexes or single-stranded oligonucleotides could not be detected by EMSA. However, duplexes with 5'-overhangs, flaps and pseudo-Y structures showed decreased mobility with added T5 exonuclease. On binding to DNA the wild-type enzyme was rendered partially resistant to proteolysis, yielding a biologically active 31.5 kDa fragment. However, the protein-DNA complex remained susceptible to inactivation by p-hydroxymercuribenzoate (PHMB, a cysteine-specific modifying agent), suggesting that neither cysteine is intimately associated with substrate binding. Replacement of both cysteine residues of the molecule with serine did not greatly alter the catalytic or binding characteristics of the protein but did render it highly resistant to inhibition by PHMB.

Original languageEnglish (US)
Pages (from-to)3801-3807
Number of pages7
JournalNucleic Acids Research
Volume25
Issue number19
DOIs
StatePublished - Oct 1 1997
Externally publishedYes

Fingerprint

Exonucleases
Bacteriophages
Cysteine
DNA
Electrophoretic Mobility Shift Assay
Oligonucleotides
Exodeoxyribonucleases
Serine
Proteolysis
Carrier Proteins
Metals
Enzymes
Proteins

ASJC Scopus subject areas

  • Genetics

Cite this

Structure-specific DNA binding by bacteriophage T5 5'→3' exonuclease. / Garforth, Scott J.; Sayers, Jon R.

In: Nucleic Acids Research, Vol. 25, No. 19, 01.10.1997, p. 3801-3807.

Research output: Contribution to journalArticle

@article{79f1e12ebcad4d61b5f64fda99a55f3e,
title = "Structure-specific DNA binding by bacteriophage T5 5'→3' exonuclease",
abstract = "Phage T5 exonuclease is a 5'→3' exodeoxyribonuclease that also exhibits endonucleolytic activity on flap structures (branched duplex DNA containing a free single-stranded 5'-end). Oligonucleotides were used to construct duplexes with either blunt ends, 5'-overhangs, 3'-overhangs, a flap or a forked end (pseudo-Y). The binding of T5 exonuclease to various structures was investigated using native electrophoretic mobility shift assays (EMSA) in the absence of the essential divalent metal cofactor. Binding of T5 exonuclease to either blunt-ended duplexes or single-stranded oligonucleotides could not be detected by EMSA. However, duplexes with 5'-overhangs, flaps and pseudo-Y structures showed decreased mobility with added T5 exonuclease. On binding to DNA the wild-type enzyme was rendered partially resistant to proteolysis, yielding a biologically active 31.5 kDa fragment. However, the protein-DNA complex remained susceptible to inactivation by p-hydroxymercuribenzoate (PHMB, a cysteine-specific modifying agent), suggesting that neither cysteine is intimately associated with substrate binding. Replacement of both cysteine residues of the molecule with serine did not greatly alter the catalytic or binding characteristics of the protein but did render it highly resistant to inhibition by PHMB.",
author = "Garforth, {Scott J.} and Sayers, {Jon R.}",
year = "1997",
month = "10",
day = "1",
doi = "10.1093/nar/25.19.3801",
language = "English (US)",
volume = "25",
pages = "3801--3807",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "19",

}

TY - JOUR

T1 - Structure-specific DNA binding by bacteriophage T5 5'→3' exonuclease

AU - Garforth, Scott J.

AU - Sayers, Jon R.

PY - 1997/10/1

Y1 - 1997/10/1

N2 - Phage T5 exonuclease is a 5'→3' exodeoxyribonuclease that also exhibits endonucleolytic activity on flap structures (branched duplex DNA containing a free single-stranded 5'-end). Oligonucleotides were used to construct duplexes with either blunt ends, 5'-overhangs, 3'-overhangs, a flap or a forked end (pseudo-Y). The binding of T5 exonuclease to various structures was investigated using native electrophoretic mobility shift assays (EMSA) in the absence of the essential divalent metal cofactor. Binding of T5 exonuclease to either blunt-ended duplexes or single-stranded oligonucleotides could not be detected by EMSA. However, duplexes with 5'-overhangs, flaps and pseudo-Y structures showed decreased mobility with added T5 exonuclease. On binding to DNA the wild-type enzyme was rendered partially resistant to proteolysis, yielding a biologically active 31.5 kDa fragment. However, the protein-DNA complex remained susceptible to inactivation by p-hydroxymercuribenzoate (PHMB, a cysteine-specific modifying agent), suggesting that neither cysteine is intimately associated with substrate binding. Replacement of both cysteine residues of the molecule with serine did not greatly alter the catalytic or binding characteristics of the protein but did render it highly resistant to inhibition by PHMB.

AB - Phage T5 exonuclease is a 5'→3' exodeoxyribonuclease that also exhibits endonucleolytic activity on flap structures (branched duplex DNA containing a free single-stranded 5'-end). Oligonucleotides were used to construct duplexes with either blunt ends, 5'-overhangs, 3'-overhangs, a flap or a forked end (pseudo-Y). The binding of T5 exonuclease to various structures was investigated using native electrophoretic mobility shift assays (EMSA) in the absence of the essential divalent metal cofactor. Binding of T5 exonuclease to either blunt-ended duplexes or single-stranded oligonucleotides could not be detected by EMSA. However, duplexes with 5'-overhangs, flaps and pseudo-Y structures showed decreased mobility with added T5 exonuclease. On binding to DNA the wild-type enzyme was rendered partially resistant to proteolysis, yielding a biologically active 31.5 kDa fragment. However, the protein-DNA complex remained susceptible to inactivation by p-hydroxymercuribenzoate (PHMB, a cysteine-specific modifying agent), suggesting that neither cysteine is intimately associated with substrate binding. Replacement of both cysteine residues of the molecule with serine did not greatly alter the catalytic or binding characteristics of the protein but did render it highly resistant to inhibition by PHMB.

UR - http://www.scopus.com/inward/record.url?scp=0030922798&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030922798&partnerID=8YFLogxK

U2 - 10.1093/nar/25.19.3801

DO - 10.1093/nar/25.19.3801

M3 - Article

C2 - 9380501

AN - SCOPUS:0030922798

VL - 25

SP - 3801

EP - 3807

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 19

ER -