| Abstract: | The ability of the SV40 large tumor antigen (T antigen), a DNA helicase, to bind to model DNA replication forks was tested.
DNA fork molecules were constructed either from two partially complementary oligonucleotides or from a single oligonucleotide
able to form a ‘panhandle’ structure. T antigen specifically recognized the two-strand fork in a reaction dependent on the
presence of ATP, dATP, or non-hydrolyzable analogs of ATP. T antigen asymmetrically bound the two-strand fork, protecting
from nuclease cleavage a fork-proximal region on only one of the two strands. The asymmetric binding is consistent with the
3′⇌5′ directionality of the DNA helicase activity of T antigen. An analogous region on the one-strand fork was also bound
by T antigen, suggesting that T antigen does not require a free singlestranded end to load onto the fork. Use of chemically
modified DNA substrates indicated that T antigen binding to the fork utilized important contacts with the DNA sugar-phosphate
backbone.
Research was supported by NIH grant AI29963, the Pew Biomedical Scholars Program (T88-00457-063), and Kaplan Cancer Center
Developmental Funding and Cancer Center Support Core Grant (from NCI P30CA16087). |
