Three temperature-sensitive (ts) mutants of simian virus 40 (SV40) in complementation group A (tsA7, tsA28, tsA30) have been isolated and characterized in permissive and restrictive host cells. At 41 C in the AH line of African green monkey kidney cells, the mutants are deficient in an early function required to produce infectious viral deoxyribonucleic acid (DNA). Temperature-shift experiments and analysis of SV40 viral DNA replication by gel electrophoresis have provided strong evidence that the ts gene product of the three mutants is directly required to initiate each new round of viral DNA replication but is not required to complete a cycle which has already begun. The synthesis of mutant DNA molecules themselves can be initiated by a nonrmutant gene product in viral complementation studies at 41 C. The cell, however, cannot substitute a host function to provide the initiator required for the replication of free viral DNA. The viral initiator is also required to establish the stable transformation of 3T3 cells.

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We have investigated the DNA-binding, oligomerization, and trans-activation functions of isolated segments of murine p53. We find that p53 has two autonomous DNA-binding regions. One domain, from amino acid 280 to 390, forms stable tetramers and binds DNA nonspecifically. The biological significance, ff any, of this DNA-binding activity is not known. A second domain, from amino acid 80 to 290, does not form stable tetramers under stringent conditions but binds DNA both specifically and nonspecifically. The specific DNA-binding function of p53, therefore, resides in the highly conserved central region of the protein and does not require stable tetramerization. Amino acids 1-290, which include both the specific DNA-binding domain and the amino-terminal acidic region, activate a p53-specific promoter in vivo. This finding strongly argues that the DNA-binding activity of p53 segment 80-290 is physiologically significant. The role of tetramerization in p53 function remains to be determined.

The simian virus 40 core origin of replication consists of nucleotides 5211 through 31. These 64 base pairs contain three functional domains with strict sequence requirements and two spacer regions with relaxed sequence specificity but precise positional constraints. The early domain extends for 10 contiguous base pairs between nucleotides 5211 and 5220. A 9-base pair spacer from sequences 5221 through 5229 separates the early domain from the 23-base pair central palindrome that directs the binding of T antigen. The late end of the core between nucleotides 12 and 31 also contains spacer and sequence-specific functions that are not yet completely mapped. We propose that the sequence-specific domains are interaction sites for viral and cellular proteins, determinants of DNA conformation, or both. The spacers would position these signals at required distances and rotations relative to one another.Simian virus 40 (SV40) encodes a replicon that is highly host specific. This host dependence suggests that viral DNA replication depends on direct interactions of host factors with viral proteins or DNA and that the viral replicon may resemble cellular replicons. The viral origin consists of multiple distinct elements. An essential core origin is sufficient for autonomous replication, but three flanking regulatory elements provide related functions that increase the efficiency of replication more than 20-fold (2, 7, 9-11, 13, 17, 21, 23, 30). These elements are T-antigen-binding region I, the 21-base pair (bp) repeats, and the 72-bp enhancers. Each ancillary region is a component of the early promoteroperator that overlaps the core origin, and each may play a role in the organization of the open region of SV40 chromatin (4,13,14,17,19,33). A single viral protein, T antigen, binds to the core origin of replication to initiate each round of DNA synthesis (8,18,23,31,37). Cellular DNA polymerase alpha is also required for replication (9). The recent development of cell-free systems for accurate initiation of viral replication opens the way for identification of additional cellular proteins that interact with the viral replicon (22,29,38).