Nucleic Acids Research, Vol 27, Issue 3 730-735, Copyright © 1999 by Oxford University Press
TJ Pickering, SJ Garforth, SJ Thorpe, JR Sayers and JA Grasby
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
endo-nucleolytic 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 10(15). 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.
ARTICLES
A single cleavage assay for T5 5'-->3' exonuclease: determination of the catalytic parameters forwild-type and mutant proteins
Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, UK.
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