Subunit structure of a yeast site-specific endodeoxyribonuclease, endo SceI. A study using monoclonal antibodies.
Endo SceI is a eucaryotic site-specific endoDNase of 120 kDa that causes double-stranded scission at well-defined sites, but is distinguishable from procaryotic restriction endonucleases by its mode of sequence recognition and lack of related specific DNA modification. In purified preparations of endoSceI, only two polypeptide species of 75 kDa (75-kDa peptide) and 50 kDa (50-kDa peptide) are detected in apparently equal amounts. We prepared mouse monoclonal IgGs that bound specifically to the 75-kDa peptide (but not the 50-kDa peptide) without inhibiting the endoSceI activity. Immunoprecipitation experiments with these IgGs revealed that the 75-kDa peptide and the 50-kDa peptide are physically associated with each other and with the endonucleolytic activity. Full endoSceI activity was recovered by mixing the purified 75-kDa peptide and the partially purified 50-kDa peptide, each of which exhibited little or no endonuclease activity alone. These observations indicate that endoSceI consists of two non-identical subunits of 75 kDa and 50 kDa, and that both subunits are required for full enzyme activity.
Nakagawa
K
K
Laboratory of Microbiology, Riken Institute, Saitama, Japan.
Hashikawa
J
J
Makino
O
O
Ando
T
T
Shibata
T
T
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D011232:Chemical Precipitation; D004262:DNA Restriction Enzymes / Q000276:immunology* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific; D004706:Endodeoxyribonucleases / Q000276:immunology* / Q000378:metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D029701:Saccharomyces cerevisiae Proteins; D004706:Endodeoxyribonucleases; D004262:DNA Restriction Enzymes; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific