BIOCHEMICAL-ANALYSIS OF MUTANTS WITH CHANGES IN THE ORIGIN-BINDING DOMAIN OF SIMIAN VIRUS-40 TUMOR-ANTIGEN

The role of the origin-binding domain of simian virus 40 large tumor a ntigen (T antigen) in the initiation of virus DNA replication was inve stigated by analyzing the biochemical activities of a series of mutant s with single-site substitutions in this region. These activities incl ude origin-specific and nonspecific DNA binding, melting of the imperf ect palindromic sequence, untwisting of the AT-rich region, unwinding of origin-containing DNA, helicase activity, and the ability to oligom erize normally in response to ATP. Three classes of T-antigen mutants that are unable to support virus replication in monkey cells are descr ibed. Class 1 mutants are unable to bind to the origin of DNA replicat ion but are able to bind to DNA nonspecifically. Class 2 mutants exhib it defective binding to both types of DNA. As expected, mutants in the se first two classes are unable to unwind origin DNA. Surprisingly, ho wever, these mutants possess significant levels of melting and untwist ing activities, suggesting that these reactions may not be solely depe ndent on the ability of the protein to recognize origin sequences. Mos t class 1 mutants oligomerize normally in response to ATP, indicating that their DNA-binding defects are not due to structural alterations b ut probably to a failure to directly recognize origin sequences. In co ntrast, class 2 mutants exhibit defective oligomerization. Class 3 mut ants bind to origin and nonorigin DNA at near wild-type levels and mel t and untwist origin DNA normally but exhibit defective oligomerizatio n and unwinding. These mutants are, however, perfectly able to carry o ut the helicase reaction, indicating that their unwinding defect is at some step after melting but before a nonspecific helicase is used to separate parental strands during replication. These results therefore suggest that proper oligomerization to correctly position the molecule s on the DNA may be more important in initiating unwinding than in bri nging about efficient DNA binding, inducing structural changes in the DNA, or carrying out the helicase reaction.