Substitutions in conserved dodecapeptide motifs that uncouple the DNA binding and DNA cleavage activities of PI-SceI endonuclease.
The PI-SceI endonuclease from yeast belongs to a protein family whose members contain two conserved dodecapeptide motifs within their primary sequences. The function of two acidic residues within these motifs, Asp218 and Asp326, was examined by substituting alanine, asparagine, and glutamic acid residues at these positions. All of the purified mutant proteins bind to the PI-SceI recognition site with the same affinity and specificity as the wild-type enzyme. By contrast, substituting alanine or asparagine amino acids at the two positions completely eliminates strand cleavage of substrate DNA, whereas substitution with glutamic acid markedly reduces the cleavage activity. Experiments using nicked substrates demonstrate that the wild-type enzyme shows no strand preference during cleavage. These results are consistent with a model in which both acidic residues are part of a single catalytic center that cleaves both DNA strands. Furthermore, substrate binding by wild-type PI-SceI stimulates hydroxyl radical or hydroxide ion attack at the cleavage site while binding by the alanine-substituted proteins either stimulates this attack significantly less or protects the DNA at this position. These finding are discussed in terms of possible reaction mechanisms for PI-SceI-mediated endonucleolytic cleavage.
Gimble
F S
FS
Center for Macromolecular Design, Texas A & M University, Houston 77030.
Stephens
B W
BW
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004706:Endodeoxyribonucleases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D017665:Hydroxyl Radical / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides / Q000737:chemistry; D010957:Plasmids; D017354:Point Mutation; D006180:Proton-Translocating ATPases; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D029701:Saccharomyces cerevisiae Proteins; D001224:Aspartic Acid; D017665:Hydroxyl Radical; D004247:DNA; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I; D006180:Proton-Translocating ATPases; C081687:VMA1 protein, S cerevisiae