ASSEMBLY OF T-ANTIGEN DOUBLE HEXAMERS ON THE SIMIAN VIRUS 40 CORE ORIGIN REQUIRES ONLY A SUBSET OF THE AVAILABLE BINDING SITES

The protein-DNA interactions that take place at eukaryotic origins of DNA replication are poorly characterized. This situation reflects, in part, the failure to identify DNA sequences that constitute higher eukaryotic origins of replication (13). Moreover, there is limited structural information about the initiator proteins that recognize origins of replication (35, 67). Indeed, structural information is currently limited to the origin binding domains of initiators encoded by simian virus 40 (SV40) (45), bovine papillomavirus (33), and Epstein-Barr virus (1, 2). In view of these limitations, a useful model for studies of the protein-DNA interactions that take place at eukaryotic origins is the binding of the virus-encoded T-antigen (T-ag) to the SV40 origin of replication. The well-characterized SV40 core origin is 64 bp long and consists of three separate domains (20, 21). The central region, termed site II (or pentanucleotide palindrome), contains four GAGGC pentanucleotides that serve as binding sites for T-ag (24, 69, 70). Site II is flanked by a 17-bp adenine-thymine (AT)-rich domain and the early palindrome (EP) (reviewed in references 4 and 29). T-ag, a 708-amino-acid phosphoprotein, has been extensively studied (reviewed in references 4, 9, and 29). The structure of the T-ag domain that is necessary and sufficient for binding to the SV40 origin, T-ag-obd131-260, was solved by use of nuclear magnetic resonance techniques (45). When the structure was viewed in terms of previous mutagenesis studies of T-ag (65, 76), considerable insight into the mechanism of binding of T-ag to individual pentanucleotides was obtained. For example, it is now apparent that site-specific binding is mediated by a pair of loops (45), a common motif in protein-DNA interactions (9, 41). Additional insights into T-ag binding to individual pentanucleotides, based on the T-ag-obd131-260 structure, were described in a recent review (9). Related studies have provided additional insights into the T-ag–core interaction; for example, studies have demonstrated that T-ag residues 121 to 135 are important for interactions with the AT-rich region (14). The initiation of SV40 replication depends upon not one but multiple protein-DNA interactions at the SV40 core origin. Initial studies demonstrated that in the absence of ATP, T-ag formed oligomers on the SV40 origin (8, 30, 31, 55). In subsequent studies, it was observed that the addition of ATP stimulated the binding of T-ag to the core origin 10- to 15-fold and induced the formation of a larger multimeric complex (6, 18, 22). With electron microscopy, it was determined that the multimeric complex was a bilobed structure (18, 19) and that each lobe contained six monomers of T-ag (48, 62). Models for double hexamer formation on the SV40 core origin have been proposed (4, 15, 48, 51, 58, 59), although they are based on limited experimental evidence. Regarding the role of the pentanucleotides in origin recognition and initiation of replication, it is known that all four pentanucleotides are necessary for origin-dependent DNA unwinding (16, 58) and replication events (16). Furthermore, most previous studies have suggested that all four pentanucleotides are required for double-hexamer formation (for reviews, see references 4, 9, and 29). However, recent studies demonstrated that all four pentanucleotides were not simultaneously bound by T-ag-obd131-260 (37). Instead, these experiments demonstrated that only two of the four pentanucleotides were required for stable binding to the core origin. Additional studies indicated that only those pentanucleotides arranged in a head-to-head orientation and separated by about one turn of a DNA double helix could serve as binding sites for T-ag-obd131-260 (37). It was proposed that T-ag-obd131-260 binding to particular pairs of pentanucleotides may mimic the nucleation of T-ag double-hexamer formation (37). In view of these studies, we elected to examine the role of the pentanucleotides in the T-ag oligomerization process. The results of these experiments are presented here.