A single cleavage assay for T5 5'→3' exonuclease

Determination of the catalytic parameters for wild-type and mutant proteins

Timothy J. Pickering, Scott J. Garforth, Simon J. Thorpe, Jon R. Sayers, Jane A. Grasby

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Bacteriophage T5 5'→3' exonuclease is a member of a family of sequence related 5'-nucleases which play an essential role in DNA replication. The 5'-nucleases have both exonucleolytic and structure-specific endonucleolytic DNA cleavage activity and are conserved in organisms as diverse as bacteriophage and mammals. Here, we report the development of a structure-specific single cleavage assay for this enzyme which uses a 5'-overhanging hairpin substrate. The products of DNA hydrolysis are characterised by mass spectrometry. The steady-state catalytic parameters of the enzyme are reported and it is concluded that T5 5'→3' exonuclease accelerates the cleavage of a specific phosphodiester bond by a factor of at least 1015. The catalytic assay has been extended to three mutants of T5 5'→3' exonuclease, K83A, K196A and K215A. Mutation of any of these three lysine residues to alanine is detrimental to catalytic efficiency. All three lysines contribute to ground state binding of the substrate. In addition, K83 plays a significant role in the chemical reaction catalysed by this enzyme. Possible roles for mutated lysine residues are discussed.

Original languageEnglish (US)
Pages (from-to)730-735
Number of pages6
JournalNucleic Acids Research
Volume27
Issue number3
DOIs
StatePublished - Feb 1 1999

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Exonucleases
Mutant Proteins
Lysine
Bacteriophages
DNA Cleavage
Enzyme Assays
Enzymes
DNA Replication
Alanine
Mammals
Mass Spectrometry
Hydrolysis
Mutation
DNA

ASJC Scopus subject areas

  • Genetics

Cite this

A single cleavage assay for T5 5'→3' exonuclease : Determination of the catalytic parameters for wild-type and mutant proteins. / Pickering, Timothy J.; Garforth, Scott J.; Thorpe, Simon J.; Sayers, Jon R.; Grasby, Jane A.

In: Nucleic Acids Research, Vol. 27, No. 3, 01.02.1999, p. 730-735.

Research output: Contribution to journalArticle

Pickering, Timothy J. ; Garforth, Scott J. ; Thorpe, Simon J. ; Sayers, Jon R. ; Grasby, Jane A. / A single cleavage assay for T5 5'→3' exonuclease : Determination of the catalytic parameters for wild-type and mutant proteins. In: Nucleic Acids Research. 1999 ; Vol. 27, No. 3. pp. 730-735.
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