Assembly of T-antigen double hexamers on the simian virus 40 core origin requires only a subset of the available binding sites.
Initiation of simian virus 40 (SV40) DNA replication is dependent upon the assembly of two T-antigen (T-ag) hexamers on the SV40 core origin. To further define the oligomerization mechanism, the pentanucleotide requirements for T-ag assembly were investigated. Here, we demonstrate that individual pentanucleotides support hexamer formation, while particular pairs of pentanucleotides suffice for the assembly of T-ag double hexamers. Related studies demonstrate that T-ag double hexamers formed on "active pairs" of pentanucleotides catalyze a set of previously described structural distortions within the core origin. For the four-pentanucleotide-containing wild-type SV40 core origin, footprinting experiments indicate that T-ag double hexamers prefer to bind to pentanucleotides 1 and 3. Collectively, these experiments demonstrate that only two of the four pentanucleotides in the core origin are necessary for T-ag assembly and the induction of structural changes in the core origin. Since all four pentanucleotides in the wild-type origin are necessary for extensive DNA unwinding, we concluded that the second pair of pentanucleotides is required at a step subsequent to the initial assembly process.
Joo
W S
WS
Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Kim
H Y
HY
Purviance
J D
JD
Sreekumar
K R
KR
Bullock
P A
PA
D000957:Antigens, Viral, Tumor / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D004261:DNA Replication; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D018741:Replication Origin; D013539:Simian virus 40 / Q000235:genetics*
D000957:Antigens, Viral, Tumor