Topoisomerase I Associates Specifically with Simian Virus 40 Large-T-Antigen Double Hexamer–Origin Complexes

Abstract

Topoisomerase I (topo I) is required for releasing torsional stress during simian virus 40 (SV40) DNA replication. Recently, it has been demonstrated that topo I participates in initiation of replication as well as in elongation. Although T antigen and topo I can bind to one another in vitro, there is no direct evidence that topo I is a component of the replication initiation complex. We demonstrate in this report that topo I associates with T-antigen double hexamers bound to SV40 origin DNA (T DH ) but not to single hexamers. This association has the same nucleotide and DNA requirements as those for the formation of double hexamers on DNA. Interestingly, topo I prefers to bind to fully formed T DH complexes over other oligomerized forms of T antigen associated with the origin. High ratios of topo I to origin DNA destabilize T DH . The partial unwinding of a smallcircular-DNA substrate is dependent on the presence of both T antigen and topo I but is inhibited at high topo I concentrations. Competition experiments with a topo I-binding fragment of T antigen indicate that an interaction between T antigen and topo I occurs during the unwinding reaction. We propose that topo I is recruited to the initiation complex after the assembly of T DH and before unwinding to facilitate DNA replication.The mechanism of initiation of eukaryotic DNA replication is not yet clearly understood. To study this process, currently the best model systems are those of simian virus 40 (SV40) and other small DNA tumor viruses. SV40 DNA replication initiates from a well-defined single origin. The core of the origin consists of three parts, a central region known as site II (which consists of four GAGGC pentanucleotide repeats), an AT-rich track, and an early palindrome (EP) region (14). This 64-bplong core is sufficient for SV40 DNA replication (15), but the efficiency of replication is enhanced by auxiliary regions on both sides of the core, especially in vivo (23).The large tumor (T) antigen is the only viral protein essential for SV40 DNA replication, while the host cells provide all other required factors (33,34,56,62). The initiation of SV40 DNA replication is a multistep event. In the presence of ATP, T antigen specifically interacts with the core of the origin and assembles into a double-hexamer structure (T DH ) (12,30,36,61). This causes partial melting of the EP region and untwisting at the AT track of the origin (3,4,5,7,13,45,47). This T DH complex appears to be the basic frame around which the replication initiation complex forms, and T DH is the functional helicase during elongation (53,54,61).At least 10 cellular proteins have been identified to be essential for complete replication of SV40 DNA (33,34,56,62). Among them, DNA polymerase ␣/primase, replication protein A (RPA), and topoisomerase I (topo I) are believed to participate in DNA replication at a very early stage (19,21,37,40,41,51,57,59,63,64,65,67). Topo I is a critical enzyme needed to release the topological stress created by DNA unwinding. RPA is required to stab...

Induction of the Bovine Papillomavirus Origin “Onion Skin”-Type DNA Replication at High E1 Protein Concentrations In Vivo

“…It is logical that the first leading replication fork on the covalently closed circular plasmid has a lower elongation rate due to the torsional stress. This stress can be elevated, for example, by the lack of topoisomerase I at the replication fork due to the squelching of topoisomerase I by the excess of E1 protein (such an interaction between E1 and topoisomerase I can occur, as has been shown for large T antigen and topoisomerase I [17]). The replication forks initiated on the newly synthesized double-stranded DNA have no such constraints and can easily catch up with the leading fork.…”

In the Simian Virus 40 In Vitro Replication System, Start Site Selection by the Polymerase α-Primase Complex Is Not Significantly Altered by Changes in the Concentration of Ribonucleotides

“…Upon forming a double hexamer on the SV40 core origin, T-ag unwinds the origin and recruits additional factors necessary for initiation (e.g., Topo I [33] and HSSB [RPA] [53; reviewed in reference 11]) and the Pol ␣-primase complex (28,52,63,75; reference 82 and references therein). Once a 3Ј-NT-5Ј or 3Ј-NTT-5Ј initiation signal is encountered by the Pol ␣-primase complex, synthesis of a primer-RNA/DNA molecule is initiated.…”

Initiation of DNA Replication from Closed Circular DNA

“…The stoichiometry of topo I to T antigen in the double hexamer is very close to 1:6 (Gai, Roy et al 2000;Simmons, Gai et al 2004), that is, one molecule of topo I per hexamer. It has been demonstrated that DNA flanking the core origin on both sides is necessary for efficient recruitment of topo I (Gai, Roy et al 2000). The most obvious mechanism of topo I recruitment is therefore that one molecule first binds to the DNA on each side of the origin.…”

“…Nevertheless, the data are convincing that both sites are needed for efficient DNA replication. If true, the stoichiometry of topo I to T antigen in the initiation complex should be 2 per hexamer, which is double the observed ratio (Gai, Roy et al 2000;Simmons, Gai et al 2004). There are several possible solutions to this dilemma.…”

“…11). Only double hexamers associated with DNA can recruit topo I (Gai, Roy et al 2000). Single hexamers and intermediate structures are inactive.…”