The CCAAT displacement protein, the homolog of the Drosophila melanogaster
CUT protein, contains four DNA-binding domains: three CUT repeats (CR1, CR2
, and CR3) and the CUT homeodomain (HD). Using a panel of fusion proteins,
we found that a CUT repeat cannot bind to DNA as a monomer, but that certai
n combinations of domains exhibit high DNA-binding affinity: CR1+2, CR3HD,
CR1HD, and CR2HD. One combination (CR1+2) exhibited strikingly different DN
A-binding kinetics and specificities. CR1+2 displayed rapid on and off rate
s and bound preferably to two C(A/G)AT sites, organized as direct or invert
ed repeats. Accordingly, only CR1+2 was able to bind to the CCAAT sequence,
and its affinity was increased by the presence of a C(A/G)AT site at close
proximity. A purified CCAAT displacement protein/CUT protein exhibited DNA
-binding properties similar to those of CR1+2; and in nuclear extracts, the
CCAAT displacement activity also required the simultaneous presence of a C
(A/G)AT site. Moreover, CR1+2, but not CR3HD, was able to displace nuclear
factor Y, Thus, the CCAAT displacement activity requires the presence of an
additional sequence (CAAT or CGAT) and involves CR1 and CR2, but not the C
UT homeodomain.