| This dissertation examines the structural changes in T antigen resulting from the binding of ATP, and its functional importance in SV40 DNA replication. In part I, analysis of carboxy-terminal truncated T antigen proteins established that the region carboxy-terminal to residue 627 was not required for oligomerization.;In part II, correlations between defects in oligomerization of single-site substitution mutations in the DNA-binding region and defects in certain biochemical activities associated with viral DNA replication were determined. Six different classes were examined. Interestingly, mutants in class 3 were unable to oligomerize properly, but were normal in origin DNA binding, melting and untwisting origin DNA, and helicase activity. However, they were defective in unwinding origin-containing DNA, suggesting that proper oligomerization to correctly position the molecules on the DNA may be more crucial in initiating unwinding than in these other activities. The identification of seven mutations in the origin-binding domain of T antigen affecting ATP-dependent oligomerization established the importance of this region of the protein for normal oligomerization.;In part III, the role of ATP-dependent oligomerization in origin unwinding was investigated by examining the ability of WT T antigen and the class 3 mutants to bind to and unwind various DNA substrates encountered during origin unwinding. Binding to all of these substrates by WT T antigen was enhanced by ATP. However, the class 3 mutants were defective in ATP-enhanced binding to nonspecific double-stranded DNA but not to single-stranded DNA. These data indicate that nonspecific double-stranded DNA binding by WT T antigen is important for opening the replication origin. Further, these data suggest that a cycle of ATP binding and ATP hydrolysis associated with enhanced and relaxed DNA binding respectively, provides a mechanism for propelling the DNA through the double-hexameric T antigen complex as unwinding proceeds.;In part IV, WT T antigen and the class 3 mutants were examined by circular dichroism. WT T antigen exhibited changes in the CD spectra in the presence of ATP, but these changes were absent in the class 3 mutants, confirming their structural abnormalities. |
