NUCLEOLIN IS A MATRIX ATTACHMENT REGION DNA-BINDING PROTEIN THAT SPECIFICALLY RECOGNIZES A REGION WITH HIGH BASE-UNPAIRING POTENTIAL

A DNA affinity column containing a synthetic double-stranded nuclear m atrix attachment region (MAR) was used to purify a 100-kDa protein fro m human erythroleukemia K562 cells. This protein was identified as nuc leolin, the key nucleolar protein of dividing cells, which is thought to control rRNA gene transcription and ribosome assembly. Nucleolin is known to bind RNA and single-stranded DNA. We report here that nucleo lin is also a MAR-binding protein. It binds double-stranded MARs from different species with high affinity. Nucleolin effectively distinguis hes between a double-stranded wild-type synthetic MAR sequence with a high base-unpairing potential and its mutated version that has lost th e unpairing capability but is still A+T rich. Thus, nucleolin is not m erely an A+T-rich sequence-binding protein but specifically binds the base-unpairing region of MARs. This binding specificity is similar to that of the previously cloned tissue-specific MAR-binding protein SATB 1. Unlike SATB1, which binds only double-stranded MARs, nucleolin bind s the single-stranded T-rich strand of the synthetic MAR probe approxi mately 45-fold more efficiently than its complementary A-rich strand, which has an affinity comparable to that of the double-stranded form o f the MAR, In contrast to the high selectivity of binding to double-st randed MARs, nucleolin shows only a small but distinct sequence prefer ence for the T-rich strand of the wild-type synthetic MAR over the T-r ich strand of its mutated version. The affinity to the T-rich syntheti c MAR is severalfold higher than to its corresponding RNA and human te lomere DNA. Quantitative cellular fractionation and extraction experim ents indicate that nucleolin is present both as a soluble protein and tightly bound to the matrix, similar to other known MAR-binding protei ns.