SEQUENCE-SPECIFIC COMPLEX-FORMATION OF DNA AND A EUKARYOTIC SEQUENCE-SPECIFIC ENDONUCLEASE, SCEI

Endo . SceI is a eukaryotic sequence-specific endonuclease of 120 kDa that causes sequence-specific double-stranded scission of DNA. Unlike results with restriction enzymes, we found a consensus sequence around the cleavage sites for Endo - SceI instead of a common sequence. We searched for conditions for studying the binding of Endo . SceI to DNA other than cutting. Under optimized conditions including gel mobility shift assay, Endo . SceI exhibited sequence-specific binding to a short double-stranded DNA (41 base pairs) containing a cleavage site and the DNA reisolated from the protein-DNA complex was not cleaved. The analysis of the complex of Endo . SceI and DNA isolated by the gel mobility shift experiments showed that the DNA-binding entity in the Endo . SceI preparation does have Endo . SceI activity and consists of an equal amount of 75-kDa and 50-kDa polypeptides. Based on this observation and those from previous studies, we conclude that Endo . SceI is a heterodimer of the 75-kDa and 50-kDa subunits. Under the present assay conditions, Endo . SceI did not show binding to single-stranded DNA having the same sequence of either plus or minus strand of the double-stranded DNA containing the cleavage site (the 41-bp DNA). Endo . SceI showed significantly higher affinity for the consensus sequence than the major cleavage site in pBR322 DNA. Unlike the cleavage of DNA by Endo . SceI which requires Mg2+, this sequence-specific binding is independent of but stimulated by Mg".