T4 endonuclease VII selects and alters the structure of the four-way DNA junction; binding of a resolution-defective mutant enzyme.
Bacteriophage T4 endonuclease VII is a nuclease that is selective for four-way DNA junctions and related branched DNA species. Using site-directed mutagenesis we have isolated a mutant protein (E86A) that is inactive in the cleavage of DNA junctions while retaining full selectivity of binding. Using endonuclease VII E86A we have shown: (1) The protein binds as a dimer to DNA junctions, with rapid exchange of subunits in free solution. (2) Binding to junctions is highly selective for the structure of DNA junctions; the complex is not displaced by a 1000-fold excess of duplex competitor DNA. (3) On binding endonuclease VII E86A to junctions, the configuration of the helical arms is significantly altered to a structure that is independent of the presence or absence of metal ions. We suggest a model for the structure of the junction in the protein complex. (4) The protein can bind to the junction in two stereochemically equivalent ways, depending upon the sequence of the junction. T4 endonuclease VII is a junction-selective enzyme that both recognises and manipulates the structure of its substrate.
Pöhler
J R
JR
CRC Nucleic Acid Structure Research Group, Department of Biochemistry, University, Dundee, UK.
Giraud-Panis
M J
MJ
Lilley
D M
DM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013205:Staphylococcal Protein A; D015032:Zinc / Q000032:analysis
D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D004247:DNA; D004706:Endodeoxyribonucleases; C035571:endodeoxyribonuclease VII; D015951:Factor Xa; D015032:Zinc