Nucleic Acids Research, Vol 25, Issue 19 3801-3807, Copyright © 1997 by Oxford University Press
SJ Garforth and JR Sayers
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.
ARTICLES
Structure-specific DNA binding by bacteriophage T5 5'-->3' exonuclease
Department of Molecular and Genetic Medicine, University of Sheffield, Royal Hallamshire Hospital, Sheffield S10 2JF, UK.
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