Evidence for DNA binding activity of numatrin (B23), a cell cycle-regulated nuclear matrix protein.
Stimulation of various cell types with growth factors is associated with a rapid induction in the synthesis of a nuclear matrix protein, termed 'numatrin' which was shown to be identical to the nucleolar protein B23. The abundance of numatrin was shown to be correlated with entry and progression through the S-phase. Thus, experiments were undertaken to examine whether numatrin also has DNA binding activity. Using whole nuclear extract, we showed that numatrin binds to both double-stranded (DS) DNA and to single-stranded (SS) DNA cellulose columns. Purified numatrin, which was extracted either under native conditions (in oligomeric form) or under urea conditions (in monomeric form), demonstrated significant binding to either [3H]DS-DNA or [3H]DS-DNA as shown by nitrocellulose filter binding assay. However, numatrin binding to DS-DNA was qualitatively and quantitatively different from its binding to SS-DNA. Thus, the binding of numatrin was several fold higher to DS-DNA as compared to SS-DNA. The binding to DS-DNA was reduced by 77% in the presence of 0.5 M NaCl, while the binding to SS-DNA was not affected under this condition. Furthermore, treatment of the native numatrin under conditions which caused monomerization of the protein resulted in a significant enhancement of numatrin binding to SS-DNA but did not affect the binding to DS-DNA. Following heparin-Sepharose chromatography purification (under native conditions), numatrin at picomole amounts showed significant binding to both DS-DNA and SS-DNA. Finally, numatrin was found to copurify with the complex of DNA polymerase alpha primase together with other proteins required for SV-40 in vitro replication activity. These results demonstrate that numatrin has DNA binding activity, and imply a possible role for numatrin/B23 in DNA-associated processes.
Feuerstein
N
N
Department of Biochemistry, University of Pennsylvania, Philadelphia.
Mond
J J
JJ
Kinchington
P R
PR
Hickey
R
R
Karjalainen Lindsberg
M L
ML
Hay
I
I
Ruyechan
W T
WT
D015153:Blotting, Western; D002453:Cell Cycle; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D019915:DNA Primase; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D000090243:Nucleophosmin; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification; D012965:Sodium Chloride / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D000090243:Nucleophosmin; D012965:Sodium Chloride; D004247:DNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases