An Alu-DNA repeat-binding protein with a molecular mass of 68 kDa (p68) is
identified in the somatic human cell nucleoplasm. Gel mobility shift assay
(GMSA), South-western blotting and affinity purification on DNA attached to
the carrier were used in the identification. GMSA revealed multiple comple
xes with the exponential dependence of their relative mobility. A narrow bi
nding site of the p68 was revealed using synthetic oligonucleotides. It is
located between the A-box and B-box of the RNA polymerase III promoter and
is identical to that reported for the Alu-binding protein from human sperma
tozoids. The same narrow binding site, the similarity of the isolation proc
edure from germ and somatic cells, and similar binding properties and molec
ular masses suggest homology of the two proteins. Antibodies raised against
Alu-protein complexes led to hypershift of the complexes in GMSA and stain
ed p68 in active fractions in human spermatozoids and in Alu-RNA-containing
alpha-RNP particles. Immunofluorescence of a HeLa cell monolayer revealed
an intranuclear dot pattern with the dots corresponding to euchromatin area
s and some dots located at the cell periphery in the cytoplasm. alpha-RNP p
articles bound Alu-DNA in vitro and contained p68 as shown using the immuno
gold procedure. Alu-DNA binding activity was revealed in cytoplasm as well
as in nucleoplasm. The possible nature of the main Alu-DNA binding protein
and its involvement in the particle structure are discussed.