T-antigen binding to site I facilitates initiation of SV40 DNA replication but does not affect bidirectionality.
SV40 origin auxiliary sequence 1 (aux-1) encompasses T-antigen (T-ag) binding site I and facilitates origin core (ori-core) activity in whole cells or cell extracts. Aux-1 activity depended completely upon its sequence, orientation and spacing relative to ori-core. Aux-1 activity was lost either by inserting 10 base pairs between aux-1 and ori-core or by placing either orientation of aux-1 on the opposite side of ori-core. Reversing the orientation of aux-1 in its normal position actually inhibited replication. Easily unwound DNA sequences that stimulate yeast or E. coli origins of replication could not replace aux-1. Aux-1 did not affect bidirectional replication. Replication remained bidirectional even when aux-1 was inactivated, and deletion of aux-1 did not affect selection of RNA-primed DNA synthesis initiation sites in the origin region: the transition from discontinuous to continuous DNA synthesis that marks the origin of bidirectional replication occurred at the same nucleotide locations in both wild-type and aux-1 deleted origins. These results support a model for initiation of SV40 DNA replication in which T-ag binding to aux-1 (T-ag binding site I) facilitates the efficiency with which T-ag initiates replication at ori-core (T-ag binding site II) without affecting the mechanism by which initiation of DNA replication occurs.
Guo
Z S
ZS
Department of Cell and Developmental Biology, Roche Research Center, Nutley, NJ 07110.
Heine
U
U
DePamphilis
M L
ML
D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004261:DNA Replication / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics*
D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral