Phosphorylation of Simian Virus 40 T Antigen on Thr 124 Selectively Promotes Double-Hexamer Formation on Subfragments of the Viral Core Origin

Initiation of DNA replication is a complicated and highly regulated process that takes place during the S phase of the cell cycle. Progress in understanding initiation events in eukaryotes includes the identification of many of the factors that catalyze nascent DNA synthesis (reviewed in references 6, 7, 19, 32 and 90). Moreover, the isolation of the origin replication complex (ORC) (2) and related factors (reviewed in references 22 and 90) has provided considerable insight into initiation of eukaryotic DNA replication. However, since origins of replication from higher eukaryotes have not been characterized (8, 19), much remains to be learned about the protein-DNA interactions that are responsible for the initiation of DNA replication in higher organisms. Experiments conducted with viral model systems have overcome certain of these limitations and aided in efforts to understand the molecular interactions that are necessary to initiate DNA replication in eukaryotes (19). In several instances, the sequences that define viral origins of replication have been established and the protein-DNA interactions that take place at these sequences have been extensively characterized (19). One particularly useful viral model system is based on simian virus 40 (SV40) DNA replication in vitro (43, 83, 95). SV40 encodes an 82-kDa protein, termed T antigen (T-ag) (84), that plays a number of critical roles during initiation of DNA replication. The functions of T-ag during the initiation of viral DNA replication have been the topic of several reviews (4, 7, 26). Briefly, T-ag site specifically binds to the SV40 origin of replication as a monomer and, as a result of a series of additional protein-protein and protein-DNA interactions (reviewed in references 4 and 7), oligomerizes into a double hexamer (13, 15, 51, 71). The double hexamer that assembles on the SV40 origin, via cooperative interactions (59, 66, 89, 93), is a functional helicase (14, 29, 80, 82, 94) that is able to unwind the SV40 origin (14, 21, 96). At the molecular level, T-ag assembly and unwinding events are poorly understood. Progress in understanding these processes includes the determination of the solution structure of the T-ag origin binding domain (OBD) (48) and images of T-ag double hexamers assembled on the SV40 origin (88). The initiation of SV40 DNA replication is highly regulated. One very important form of regulation is determined by the phosphorylation state of T-ag (for reviews, see references 24 to 26, 67, and 92). Of particular importance is phosphorylation of T-ag on threonine 124 (23, 38, 53–56, 77). Indeed, phosphorylation of T-ag on Thr 124 is the sole posttranslational modification required for origin-dependent unwinding (54) and DNA replication (53, 54, 56, 77). The enzyme that phosphorylates T-ag on Thr 124 has not been unequivocally identified (25); however, in vitro studies suggest that it is a member of the cyclin–cyclin-dependent kinase (CDK) complex (31, 53, 54, 56). In contrast to activation via Thr 124 phosphorylation, phosphorylation of serine residues 120, 123, 677, and 679 inhibits initiation of viral replication (9, 23, 30, 40, 58, 73, 75, 79, 89). Consistent with these findings, newly synthesized T-ag, phosphorylated at Thr 124 and Thr 701, has a higher affinity for SV40 DNA than older T-ag molecules that are also phosphorylated on numerous serine residues (63, 73). Models for the control of SV40 replication, via dephosphorylation of serine residues and phosphorylation of Thr 124, have been proposed (23–25, 67). A mutant T-ag molecule, containing a threonine-to-alanine substitution at position 124 (T124A), has proven to be a useful reagent for studies designed to understand the role played by Thr 124 phosphorylation during initiation of replication. T124A molecules assemble both hexamers and double hexamers on the SV40 origin, have helicase activity, distort the structure of the core origin, bind to cellular proteins required for initiation, and yet are not able to support origin unwinding or DNA synthesis (23, 54, 56, 77, 89, 93). These studies indicate that the T124A mutant is defective at some point between T-ag binding and DNA unwinding. Considerable effort has been expended in characterizing the SV40 core origin, the segment of DNA at which T-ag site specifically binds. The core origin contains three subdomains: a central region, termed site II, that is flanked by an AT-rich domain, and a second region termed the early palindrome (EP) (16, 17, 65). Site II consists of four pentanucleotides (GAGGC) arranged as inverted pairs that serve as binding sites for T-ag (18, 48, 85, 86). It has been reported that the entire core origin is not required for T-ag assembly (35, 39, 81a). Indeed, mutant origins containing single pentanucleotides support hexamer formation, while properly arranged pairs of pentanucleotides support double hexamer formation (35). Related studies demonstrated that the 64-bp core origin contains two separate assembly units for double hexamer formation. One consists of pentanucleotides 1 and 3 and the EP (called the penta 1,3 + EP assembly unit) while the second is composed of pentanucleotides 2 and 4 and the AT region (penta 2,4 + AT assembly unit) (39, 81a). Recent experiments indicate that on the penta 1,3 and EP assembly unit, the first hexamer assembles on pentanucleotide 1 and the second hexamer assembles on pentanucleotide 3. On the penta 2,4 + AT assembly unit, the first hexamer forms on pentanucleotide 4 and the second forms on pentanucleotide 2 (81a). The 64-bp core origin is known to afford alternative modes of T-ag assembly (35, 39; Sreekumar et al., unpublished). In contrast, oligonucleotides containing individual assembly units restrict the positions at which hexamers and double hexamers can assemble. This is an obvious advantage when conducting studies designed to examine how double hexamer assembly is regulated. Therefore, we have used T-ag and the T124A mutant, along with subfragments of the core origin containing single assembly units (39, 81a), to reinvestigate the role of Thr 124 phosphorylation in the regulation of T-ag assembly. Results from these studies are presented herein.