Structure-specific DNA binding by bacteriophage T5 5'-->3' exonuclease.
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.
Garforth
S J
SJ
Department of Molecular and Genetic Medicine, University of Sheffield, Royal Hallamshire Hospital, Sheffield S10 2JF, UK.
Sayers
J R
JR
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005090:Exodeoxyribonucleases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D004791:Enzyme Inhibitors; D006902:Hydroxymercuribenzoates; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C030601:4-hydroxymercuribenzoate; D004247:DNA; D005090:Exodeoxyribonucleases; C079044:T5 D15 exonuclease