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 Mg2+.
Kawasaki
K
K
Laboratory of Microbiology, Riken (Institute of Physical and Chemical Research), Wako-shi, Japan.
Takahashi
M
M
Ando
T
T
Shibata
T
T
D001483:Base Sequence; D001665:Binding Sites; D002853:Chromatography, Liquid; D004247:DNA / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific