NBP, a protein that specifically binds an enhancer of immunoglobulin gene rearrangement: purification and characterization.
Immunoglobulin and T-cell receptor (TCR) genes are encoded in discrete germ line DNA segments that are joined by site-specific recombination during lymphocyte development. These DNA rearrangements are mediated by conserved heptamer and nonamer DNA sequence elements that lie near the sites of recombination. In this paper we show that the nonamer element coincides with the recognition site for a specific DNA-binding protein: mutations within the nonamer sequence, but not outside of it, decrease affinity for the binding protein by 300- to 1000-fold. Deletion of the binding site for the protein results in at least a 50-fold decrease in recombination frequency in vivo. By a combination of conventional and recognition site affinity chromatography, we have achieved greater than 20,000-fold purification of the protein from calf thymus, with an overall yield of 22%. The purified protein, which we now call nonamer-binding protein (NBP), has an apparent molecular weight of 63,000 and a frictional ratio of 1.27, suggesting that it exists as a globular monomer in 0.5 M NaCl. Our observations suggest that NBP is a component of the recombinational apparatus.
Li
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Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Morzycka-Wroblewska
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002458:Cell Fractionation; D002460:Cell Line; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D015321:Gene Rearrangement; D005803:Genes, Immunoglobulin; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D011948:Receptors, Antigen, T-Cell / Q000235:genetics; D011995:Recombination, Genetic
D004268:DNA-Binding Proteins; D011948:Receptors, Antigen, T-Cell; D004247:DNA