BINDING, BENDING AND CLEAVAGE OF DNA SUBSTRATES BY THE HOMING ENDONUCLEASE PI-SCEI

To characterize the interaction between the homing endonuclease PI-Sce l 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 simi lar to 30 bp of the natural recognition sequence using supercoiled pla smids, similar to 40-50 bp using linearized plasmids and > 50 bp using synthetic double-stranded oligodeoxynucleotides. Cleavage of supercoi led plasmids occurs without accumulation of the nicked intermediate. I n the presence of Mn2+, DNA cleavage by PI-Scel is more efficient than with Mg2+ and already occurs with substrates containing a shorter par t 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-Scel binds w ith high affinity to one of its cleavage products, a finding which may explain why PI-Scel hardly shows enzymatic turnover in vitro. Upon bi nding, two complexes are formed, which differ in the degree of bending (45 degrees versus 75 degrees). According to a phasing analysis bendi ng is directed into the major groove. Strong binding, not, however, cl eavage is also observed with the genetically engineered enzymatically inactive variant comprising amino acids 1-277, Models for binding and cleavage of DNA by PI-Scel are discussed based on these results.