-transparent.png){kind=logo}
By performing DNase I footprint analysis, we had identified three distinct protein binding sequences (MT1, MT2, and MT3) located on the mouse thymidine kinase (TK) upstream promoter (Dou, Q.-P., Fridovich-Keil, J. L., and Pardee, A.B. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1157-1161). Here we report that MT2 includes an E2F-like binding site (GTTCGCGGGCAAA), as shown by the following evidence. (i) MT2 bound specifically to an affinity-purified fusion human E2F protein. (ii) Both MT2 and an authentic E2F site (TTTCGCGCGCTTT) bound specifically to similar or identical nuclear protein complexes. (iii) Formation of both these DNA-protein complexes were cell cycle-dependent: a G0/G1 phase-specific complex (E2F.G0/G1) was replaced by an S phase-specific complex(es) (E2F.S), whereas "free" E2F increased after the G1/S transition. (iv) Pulse inhibition of protein synthesis with cycloheximide interchanged these complexes with similar kinetics. (v) When MT2-shifted E2F.G0/G1, E2F.S, and free E2F were eluted and analyzed by Western blot assay using a specific antiserum to human E2F-1, two forms of murine E2F (62 and 66 kDa) were observed from all three complexes. The compositions of these MT2-bound complexes were also investigated. Studies using specific antibodies revealed that p107, a retinoblastoma-like protein, was present in both E2F-G0/G1 and E2F.S, whereas cyclin E.cyclin A.cdk2 were only present in E2F.S complex(es). These data suggest that removal of the p107-containing E2F.G0/G1 complex, a candidate repressor, from the MT2 site in late G1 may be essential for S phase-dependent transcription of the mouse TK gene.
Q P Dou, and
S Zhao, and
A H Levin, and
J Wang, and
K Helin, and
A B Pardee
July 1990,
Molecular and cellular biology,
Q P Dou, and
S Zhao, and
A H Levin, and
J Wang, and
K Helin, and
A B Pardee
February 1992,
The Journal of biological chemistry,
Q P Dou, and
S Zhao, and
A H Levin, and
J Wang, and
K Helin, and
A B Pardee
February 1991,
Proceedings of the National Academy of Sciences of the United States of America,
Q P Dou, and
S Zhao, and
A H Levin, and
J Wang, and
K Helin, and
A B Pardee
December 1994,
Biochemical and biophysical research communications,
Q P Dou, and
S Zhao, and
A H Levin, and
J Wang, and
K Helin, and
A B Pardee
November 1998,
Nucleic acids research,
Q P Dou, and
S Zhao, and
A H Levin, and
J Wang, and
K Helin, and
A B Pardee
April 1992,
Proceedings of the National Academy of Sciences of the United States of America,
Q P Dou, and
S Zhao, and
A H Levin, and
J Wang, and
K Helin, and
A B Pardee
September 1985,
Cell,
Q P Dou, and
S Zhao, and
A H Levin, and
J Wang, and
K Helin, and
A B Pardee
June 1995,
Journal of cellular physiology,
Q P Dou, and
S Zhao, and
A H Levin, and
J Wang, and
K Helin, and
A B Pardee
April 1993,
Proceedings of the National Academy of Sciences of the United States of America,
Q P Dou, and
S Zhao, and
A H Levin, and
J Wang, and
K Helin, and
A B Pardee
June 2004,
The Journal of biological chemistry,
