Simian virus 40 origin auxiliary sequences weakly facilitate T-antigen binding but strongly facilitate DNA unwinding.
The complete simian virus 40 (SV40) origin of DNA replication (ori) consists of a required core sequence flanked by two auxiliary sequences that together increase the rate of DNA replication in monkey cells about 25-fold. Using an extract of SV40-infected monkey cells that reproduced the effects of ori-auxiliary sequences on DNA replication, we examined the ability of ori-auxiliary sequences to facilitate binding of replication factors and to promote DNA unwinding. Although the replicationally active form of T antigen in these extracts had a strong affinity for ori-core, it had only a weak but specific affinity for ori-auxiliary sequences. Deletion of ori-auxiliary sequences reduced the affinity of ori-core for active T antigen by only 1.6-fold, consistent with the fact that saturating concentrations of T antigen in the cell extract did not reduce the stimulatory role of ori-auxiliary sequences in replication. In contrast, deletion of ori-auxiliary sequences reduced the efficiency of ori-specific, T-antigen-dependent DNA unwinding in cell extracts at least 15-fold. With only purified T antigen in the presence of topoisomerase I to unwind purified DNA, ori-auxiliary sequences strongly facilitated T-antigen-dependent DNA conformational changes consistent with melting the first 50 base pairs. Under these conditions, ori-auxiliary sequences had little effect on the binding of T antigen to DNA. Therefore, a primary role of ori-auxiliary sequences in DNA replication is to facilitate T-antigen-dependent DNA unwinding after the T-antigen preinitiation complex is bound to ori-core.
Gutierrez
C
C
Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Guo
Z S
ZS
Roberts
J
J
DePamphilis
M L
ML
D000818:Animals; D000952:Antigens, Polyomavirus Transforming; D001667:Binding, Competitive; D002460:Cell Line; D003001:Cloning, Molecular; D004265:DNA Helicases; D004261:DNA Replication; D004270:DNA, Circular / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D007700:Kinetics; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D010957:Plasmids; D015183:Restriction Mapping; D013539:Simian virus 40 / Q000235:genetics* / Q000276:immunology
D000952:Antigens, Polyomavirus Transforming; D004270:DNA, Circular; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D004265:DNA Helicases