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 previousl y demonstrated that chronic morphine treatment decreases the DNA bindi ng activity of ssCRE-BP (single-stranded cyclic AMP response element-b inding protein), which has been shown to be identical to pur alpha by cDNA cloning, and is abundant in the brain. In this report we identifi ed an activator of ssCRE-BP/pur alpha in the brain and characterized i t. Although purified ssCRE-BP/pur alpha or its GST-fusion protein exhi bited very low DNA binding activities, they were markedly enhanced by including nuclear extract in the binding assay. The enhanced binding a ctivity is trypsin-sensitive, heat-stable and has a molecular weight o f 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 f ound 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. (C) 1997 Elsevier Science Ltd.