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.

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Journal Article | Research Support, Non-U.S. Gov't

Authors

Wende W, Grindl W, Christ F, Pingoud A, Pingoud V

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