Binding, bending and cleavage of DNA substrates by the homing endonuclease Pl-SceI.
To characterize the interaction between the homing endonuclease PI-SceI and DNA, we prepared different DNA substrates containing the natural recognition sequence or parts thereof. Depending on the nature of the substrates, efficient cleavage is observed with a DNA containing approximatel 30 bp of the natural recognition sequence using supercoiled plasmids, approximately 40-50 bp using linearized plasmids and > 50 bp using synthetic double-stranded oligodeoxynucleotides. Cleavage of supercoiled plasmids occurs without accumulation of the nicked intermediate. In the presence of Mn2+, DNA cleavage by PI-SceI is more efficient than with Mg2+ and already occurs with substrates containing a shorter part of the recognition sequence. The requirements for strong binding are less stringent: a 35 bp oligodeoxynucleotide which is not cleaved is bound as firmly as other longer oligodeoxynucleotides. PI-SceI binds with high affinity to one of its cleavage products, a finding which may explain why PI-SceI hardly shows enzymatic turnover in vitro. Upon binding, two complexes are formed, which differ in the degree of bending (45 degrees versus 75 degrees). According to a phasing analysis bending is directed into the major groove. Strong binding, not, however, cleavage is also observed with the genetically engineered enzymatically inactive variant comprising amino acids 1-277. Models for binding and cleavage of DNA by PI-SceI are discussed based on these results.
Wende
W
W
Institut für Biochemie, Justus-Liebig-Universität, Giessen, Germany.
Grindl
W
W
Christ
F
F
Pingoud
A
A
Pingoud
V
V
D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011485:Protein Binding; D006180:Proton-Translocating ATPases; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D004278:DNA, Superhelical; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; D006180:Proton-Translocating ATPases; C081687:VMA1 protein, S cerevisiae