Abstract
A DNA affinity column containing a synthetic double-stranded nuclear
matrix attachment region (MAR) was used to purify a 100-kDa protein
from human erythroleukemia K562 cells. This protein was identified
as nucleolin, 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 nucleolin is also a MAR-binding protein. It binds double-stranded
MARs from different species with high affinity. Nucleolin effectively
distinguishes between a double-stranded wild-type synthetic MAR sequence
with a high base-unpairing potential and its mutated version that
has lost the unpairing capability but is still A+T rich. Thus, nucleolin
is not merely 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 SATB1. Unlike SATB1, which binds only double-stranded MARs,
nucleolin binds the single-stranded T-rich strand of the synthetic
MAR probe approximately 45-fold more efficiently than its complementary
A-rich strand, which has an affinity comparable to that of the double-stranded
form of the MAR. In contrast to the high selectivity of binding to
double-stranded MARs, nucleolin shows only a small but distinct sequence
preference for the T-rich strand of the wild-type synthetic MAR over
the T-rich strand of its mutated version. The affinity to the T-rich
synthetic MAR is severalfold higher than to its corresponding RNA
and human telomere DNA. Quantitative cellular fractionation and extraction
experiments indicate that nucleolin is present both as a soluble
protein and tightly bound to the matrix, similar to other known MAR-binding
proteins.
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