Investigation of the DNA binding properties of the simian virus 40 (SV40) A protein (large T antigen) and the hybrid adenovirus-SV40 D2 protein revealed that both viral proteins protect similar regions of SV40 DNA from digestion by DNase I or methylation by dimethyl sulfate. However, the interaction of D2 protein with DNA was more sensitive to increases of NaCl concentration than was the interaction of wild-type SV40 A protein. Dimethylsulfate footprinting identified 13 DNA pentanucleotide contact sites at the viral origin of replication. The sequences of these sites corresponded to the consensus family 5'-(G>T) (A>G)GGC-3'. The pentanucleotides were distributed in three regions of origin DNA. Region I contained three pentanucleotide contact sites arranged as direct repetitions encompassing a span of 23 base pairs. In region II, four pentanucleotides were oriented as inverted repetitions that also spanned a total of 23 base pairs. Region III had six recognition pentanucleotides arranged as direct repetitions in a space of 59 base pairs. These fundamental variations in DNA arrangement are likely to determine different patterns of protein binding in each region.

DNA binding regions I, II, and III at the origin of replication have different arrangements of A protein (T antigen) recognition pentanucleotides. The A protein also protects each region from DNase in distinctly different patterns. Footprint and fragment assays led to the following conclusions: (i) in some cases a single recognition pentanucleotide is sufficient to direct the binding and accurate alignment of A protein on DNA; (ii) the A protein binds within isolated region I or II in a sequential process leading to multiple overlapping areas of DNase protection within each region; and (iii) the 23-base pair span of recognition sequences in region II allows binding and protection of a longer length of DNA than the 23-base pair span in region I. We propose a model of protein binding that addresses the problem of variations in the arrangement of pentanucleotides in regions I and II and explains the observed DNase protection patterns. The central feature of the model requires each protomer of A protein to bind to a pentanucleotide in a unique direction. The resulting orientation of protein would protect more DNA at the 5' end of the 5'-GAGGC-3' recognition sequence than at the 3' end. The arrangement of multiple protomers at the origin of simian virus 40 replication is discussed. of PIPES binding buffer with 0.1 M MgCl2 and 0.05 M CaCl2 was added for 5 min at 0°C in 0.005 M NaCl.

Wild-type simian virus 40 A protein (large T antigen) bound to three tandem regions of simian virus 40 DNA. The binding regions were defined by the ability of A protein to protect simian virus 40 DNA from digestion with limited (footprint assay) or excess (fragment assay) amounts of DNase I. At low concentrations, protein first bound to region I, which maps 30 to 45 base pairs to the early side of the origin of replication. At higher concentrations, A protein also protected region II and then region III. Region II spanned approximately 65 base pairs and corresponded in location to the functional origin of replication that contains a unique Bgl I site along with an adjacent adenine-thymine-rich region. Region III was adjacent to the late boundary of region II, but its distal limit was not well defined. Twelve distinct temperature-sensitive ( ts ) A proteins were purified and examined for their ability to bind in regions I to III. Three classes of ts A protein were defined on the basis of thermal stability. Class I ts A protein displayed wild-type binding either with or without a heat shock. Unheated class II ts A protein exhibited wild-type binding, but after a heat shock bound very poorly to the origin of replication. Class III ts A protein was defective in its binding even without a heat shock and only protected region I. Classes II and III were coded by mutants mapping in two distinct regions of the genome. For all of the ts A proteins examined, there was a positive correlation between the thermolability of origin binding in vitro and the temperature sensitivity of these mutants for DNA replication and transcriptional autoregulation in vivo. This correlation adds support to the essential role of origin binding by A protein in viral DNA replication and early transcription repression.

Previous studies with wild-type simian virus 40 DNA have shown that the sequence 5'-GAGGC-3' directs the binding of A protein (T antigen). The functional origin of replication contains four recognition pentanucleotides each of which is separated by a single base pair and arranged as two pairs of direct repetitions that are inverted relative to each other. Analysis of A protein binding to a series of nonviable mutanfts progressively deleting these contact sites leads to the following conclusions: (i) stable binding of subunits of A protein to three origin pentanucleotides is not sufficient for the initiation of DNA replication, (ii) the stability of DNA binding depends on interactions between bound protein subunits, and (iii) a single pentanucleotide is sufficient to bind and orient a subunit of A protein.