Characterization of a nuclear factor that enhances DNA binding activity of SSCRE-BP/PUR alpha, a single-stranded DNA binding protein.
Pur alpha has been identified as a single-stranded DNA binding protein that specifically binds to the purine-rich strand present in the DNA replication initiation zone of the human c-myc gene. We have previously demonstrated that chronic morphine treatment decreases the DNA binding activity of ssCRE-BP (single-stranded cyclic AMP response element-binding protein), which has been shown to be identical to pur alpha by cDNA cloning, and is abundant in the brain. In this report we identified an activator of ssCRE-BP/pur alpha in the brain and characterized it. Although purified ssCRE-BP/pur alpha or its GST-fusion protein exhibited very low DNA binding activities, they were markedly enhanced by including nuclear extract in the binding assay. The enhanced binding activity is trypsin-sensitive, heat-stable and has a molecular weight of approximately 66 kDa. Casein could substitute for the activator and increased the DNA binding activity of ssCRE-BP/pur alpha by one order. A series of deletion mutants were prepared in order to determine the DNA binding and activator interacting domains, and both of them were found to reside in AA 50-215 of ssCRE-BP/pur alpha. These data suggest that the DNA binding activity of ssCRE-BP/pur alpha is augmented by a nuclear protein, which may modulate the ssCRE-BP/pur alpha activity to develop morphine dependence and tolerance.
Ding
Y
Y
Department of Pharmacology 1, Osaka University Medical School, Japan.
Osugi
T
T
Kuo
C H
CH
Tanaka
H
H
Do
E
E
Irie
Y
Y
Miki
N
N
D000818:Animals; D001685:Biological Factors; D002364:Caseins / Q000378:metabolism*; D002467:Cell Nucleus / Q000737:chemistry*; D002531:Cerebellum / Q000737:chemistry; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D009154:Mutation; D009419:Nerve Tissue Proteins; D017434:Protein Structure, Tertiary; D014157:Transcription Factors
D001685:Biological Factors; D002364:Caseins; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; C121485:Pura protein, mouse; D014157:Transcription Factors