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Selection of non-specific DNA cleavage sites by the Type IC restriction endonuclease EcoR124IThe Type IC restriction endonuclease EcoR124I translocates DNA prior to cleavage in an ATP-dependent manner. Thus the enzyme is able to cleave DNA distal to the recognition sequence. Any mechanism which leads to cessation of translocation can lead to the cleavage of the DNA. On linear DNA this event usually occurs when two enzymes, translocating DNA, collide leading to cleavage at a mid-point between two recognition site (Studier and Bandyopadhyay, 1988). However, on circular DNA this can occur when translocation leads to such a high degree of supercoiling such that topological restraints will halt the translocation process leading to DNA cleavage. However, we investigated the cleavage of both circular and linear DNA and discovered discrete cleavage sites within 250bp of the recognition sequence (Szczelkun et al., 1997). These cleavage sites were not confined to particular DNA sequences or DNA topology. Therefore, a model is required to account for both types of cleavage.
One possible explanation is that dissociation of one of the HsdR subunits dissociates from the endonuclease before translocation occurs. This halts translocation in one direction and leads to DNA cleavage at or near the recognition sequence. While such dissociation has been shown to be a fundamental part of the stalling process for the enzyme EcoR124I (Seidel et al 2005), it has also been shown that DNA cleavage requires a R2-complex (Janscak et al 1998) . An alternative situation is that a stalling event occurs before significant translocation and this leads to cleavage new the binding site, which seems to fit the data better. |
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