Purification of the simian virus 40 (SV40) T antigen DNA-binding domain and characterization of its interactions with the SV40 origin.
To better define protein-DNA interactions at a eukaryotic origin, the domain of simian virus 40 (SV40) large T antigen that specifically interacts with the SV40 origin has been purified and its binding to DNA has been characterized. Evidence is presented that the affinity of the purified T antigen DNA-binding domain for the SV40 origin is comparable to that of the full-length T antigen. Furthermore, stable binding of the T antigen DNA-binding domain to the SV40 origin requires pairs of pentanucleotide recognition sites separated by approximately one turn of a DNA double helix and positioned in a head-to-head orientation. Although two pairs of pentanucleotides are present in the SV40 origin, footprinting and band shift experiments indicate that binding is limited to dimer formation on a single pair of pentanucleotides. Finally, it is demonstrated that the T antigen DNA-binding domain interacts poorly with single-stranded DNA.
Joo
W S
WS
Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Luo
X
X
Denis
D
D
Kim
H Y
HY
Rainey
G J
GJ
Jones
C
C
Sreekumar
K R
KR
Bullock
P A
PA
D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D013539:Simian virus 40 / Q000276:immunology*
D000952:Antigens, Polyomavirus Transforming; D010446:Peptide Fragments; D004247:DNA