Purification and characterization of nuclear scaffold proteins which bind to a highly repetitive bent DNA from rat liver.
Our previous work (Hibino et al. (1992) Biochem. Biophys. Res. Commun. 184, 853-858) has shown that the binding affinities of a highly repetitive DNA component for rat nuclear scaffold proteins, P123 and P130, depend on the degree of sequence-directed bending of the helix axis. In the present experiment, these proteins have been purified and finally isolated by DNA-Sepharose column chromatography. The pI values of P123 and P130 were 7.2 and 8.1, respectively. The southwestern blotting revealed that a highly repetitive bent DNA (370-bp XmmI fragment) from rat liver binds readily to the isolated proteins under a hypotonic condition (50 mM NaCl) and that the level of the binding affinity for each protein was lowered with increasing NaCl concentration. The sedimentation analysis predicted that direct interaction between the XmnI fragment and P123 or P130 results in the formation of a complex which consists of two of the fragments and one molecule of the protein, alternatively, one of the fragment and three molecules of the proteins. Distamycin A, an antibiotic which binds specifically to AT-rich DNA, removed the bend in the XmnI fragment and inhibited binding of the fragment to P123 or P130, whereas neither removal of the bend nor binding inhibition was observed with chromomycin A3, an antibiotic specific for GC-rich sites in DNA. These results imply that AT-rich regions in a highly repetitive DNA component cause bending of the helix axis to be recognized by some of nuclear scaffold proteins.
Hibino
Y
Y
Cell Biology Division, Faculty of Pharmaceutical Sciences, Toyama Medical & Pharmaceutical University, Japan.
Nakamura
K
K
Tsukada
S
S
Sugano
N
N
D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008099:Liver / Q000378:metabolism*; D009687:Nuclear Proteins / Q000302:isolation & purification* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D051381:Rats; D012091:Repetitive Sequences, Nucleic Acid
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004247:DNA