Interaction of deoxyinosine 3'-endonuclease from Escherichia coli with DNA containing deoxyinosine.
By using a band mobility shift assay, deoxyinosine 3'-endonuclease, an Escherichia coli enzyme which recognizes deoxyinosine, AP site, urea residue, and base mismatches in DNA, was shown to bind tightly to deoxyinosine-containing oligonucleotide duplexes. Two distinct protein-DNA complexes were observed, the faster migrating complex (complex I, Kd = 4 x 10(-9) M) contained one molecule of deoxyinosine 3'-endonuclease, while the slower migrating complex (complex II, Kd = 4 x 10(-7) M) contained two molecules of the protein bound to every molecule of duplex DNA. The endonucleolytic activity of deoxyinosine 3'-endonuclease paralleled the formation of the complex I. Interestingly, deoxyinosine 3'-endonuclease exhibited similar affinities for both the substrate and the nicked duplex product and thus remained bound to the DNA after the cleavage reaction. The formation of a stable complex required the presence of a duplex structure 5' to the deoxyinosine residue. DNase I footprinting revealed that deoxyinosine 3'-endonuclease protected 4-5 nucleotides 5' to the deoxyinosine, and when complex II was formed, at least 13 nucleotides 3' to deoxyinosine were protected. Based on these results, a model is proposed for the interaction of deoxyinosine 3'-endonuclease with DNA containing deoxyinosine.
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007288:Inosine / Q000031:analogs & derivatives* / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D007288:Inosine; D004247:DNA; D004706:Endodeoxyribonucleases; C089421:deoxyinosine 3' endonuclease; D003850:Deoxyribonuclease I; C012271:deoxyinosine