The junction-resolving enzyme T7 endonuclease I: quaternary structure and interaction with DNA.
Endonuclease I is a DNA junction-selective resolving enzyme from bacteriophage T7. Using a nuclease-defective mutant that retains normal binding to DNA we show that the protein binds to four-way DNA junctions as a dimer, in common with other junction-resolving enzymes studied. Gel filtration and chemical crosslinking indicate that endonuclease I also exists in free solution as a dimer together with a tetramer and higher molecular mass aggregates. However, in marked contrast with other junction-resolving enzymes, there is no detectable subunit exchange under normal conditions. Only by exposure to 6 M urea could we induce subunit exchange, and this was used to generate heterodimeric species containing one active and one inactive subunit. Using a supercoil-stabilised cruciform substrate we demonstrate that an active subunit of endonuclease I can act as a junction-specific nuclease in a heterodimeric combination with an inactive subunit. However, the two subunits of a fully active homodimeric enzyme each cleave the phosphodiester backbone of a cruciform within the lifetime of the DNA-protein complex.
Parkinson
M J
MJ
Department of Biochemistry, The University of Dundee, UK.
Lilley
D M
DM
D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D011487:Protein Conformation; D011489:Protein Denaturation; D012996:Solutions; D014508:Urea / Q000378:metabolism
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D012996:Solutions; D014508:Urea; D003850:Deoxyribonuclease I