Mapping of helicase and helicase substrate-binding domains on simian virus 40 large T antigen.
We generated fragments of simian virus 40 large tumor antigen (T antigen) by tryptic digestion and assayed them for helicase activity and helicase substrate (mostly single-stranded DNA)-binding activity in order to map the domain sites on the protein. The N-terminal 130 amino acids were not required for either activity, since a 76-kilodalton (kDa) fragment (amino acids 131 to 708) was just as active as intact T antigen. To map the helicase domain further, smaller tryptic fragments were generated. A 66-kDa fragment (131 to about 616) retained some activity, whereas a slightly smaller 62-kDa fragment (137 or 155 to 616) had none. This suggests that the minimal helicase domain maps from residue 131 to approximately residue 616. To map the helicase substrate-binding domain, we tested various fragments in a substrate-binding assay. The smallest fragment for which we could clearly demonstrate activity was a 46-kDa fragment (131 to 517). To determine the relationship between the helicase substrate domain and the origin-binding domain (131 to 257, minimal core region; 131 to 371, optimal region), we performed binding experiments with competitor DNAs present. We found that origin-containing double-stranded DNA was an excellent competitor of the binding of the helicase substrate to T antigen, suggesting that the two domains overlap. Therefore, full helicase activity requires at least a partial origin-binding domain as well as an active ATPase domain. Additionally, we found that the helicase substrate was a poor competitor of origin-binding activity, indicating that T antigen has a much higher affinity to origin sequences than to the helicase substrate.
Wun-Kim
K
K
School of Life and Health Sciences, University of Delaware, Newark 19716.
Simmons
D T
DT
D000818:Animals; D000952:Antigens, Polyomavirus Transforming; D001667:Binding, Competitive; D002460:Cell Line; D004265:DNA Helicases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D008715:Methionine; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping; D013539:Simian virus 40 / Q000276:immunology*; D013379:Substrate Specificity; D014357:Trypsin
D000952:Antigens, Polyomavirus Transforming; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D008715:Methionine; D014357:Trypsin; D004265:DNA Helicases