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Addition of purified human topoisomerase I (topo I) to simian virus 40 T antigen-driven in vitro DNA replication reactions performed with topo I-deficient extracts results in a greater than 10-fold stimulation of completed molecules as well as a more than 3-fold enhancement of overall DNA replication. To further characterize this stimulation, we first demonstrate that bovine topo I but not Escherichia coli topo I can also enhance DNA replication. By using several human topo I mutants, we show that a catalytically active form of topo I is required. To delineate whether topo I influences the initiation or the elongation step of replication, we performed delayed pulse, pulse-chase, and delayed pulse-chase experiments. The results illustrate that topo I cannot promote the completion of partially replicated molecules but is needed from the beginning of the reaction to initiate replication. Competitive inhibition experiments with the topo I binding T antigen fragment 1-246T and a catalytically inactive topo I mutant suggest that part of topo I's stimulation of replication is mediated through a direct interaction with T antigen. Collectively, our data indicate that topo I enhances the synthesis of fully replicated DNA molecules by forming essential interactions with T antigen and stimulating initiation.A major area of interest under investigation is the molecular mechanism involved in the early stages of mammalian cell DNA replication. However, neither the mammalian initiator protein(s) nor the helicase(s) specific for DNA replication has been definitively identified (23,25). Moreover, the exact origins of mammalian DNA replication are also not defined and appear to reside in initiation zones rather than consist of precise DNA consensus sequences (27,29). To circumvent these obstacles and still obtain valuable information regarding the early stages of mammalian cell DNA replication, we are exploiting the simian virus 40 (SV40) model of DNA replication in vitro, which depends on a well-defined origin of replication and a single initiator protein and DNA helicase, the virally encoded T antigen (10,35,59). All essential replication machinery components except T antigen are provided by permissive cellular extracts (34,35,59,68). Further studies have led to the establishment of reconstituted systems from purified proteins that support SV40 DNA replication (26,64,65,69). Use of these model systems has provided a wealth of information regarding the functions of proteins involved in eukaryotic DNA replication (recently reviewed in references 5 and 20). However, there are still significant gaps in our understanding of the exact mechanisms involved in eukaryotic DNA replication, in particular with respect to (i) the composition and geometry of the initiation and elongation complexes, (ii) the interactions among replication factors, and (iii) how replication is regulated at various stages.T antigen provides the helicase activity by forming a double hexamer over the SV40 origin of replication in the presence of ATP (3,11,38). In a...

We have previously found that purified SV40 T antigen and topoisomerase I (topo I) bind to one another in vitro. In this report, we determined the effects of human topo I on T antigen-mediated DNA replication and investigated whether it altered T antigen's biochemical activities. Topo I stimulates DNA replication and especially increases the amounts of finished circular molecules. This protein had no effect on T antigen's ability to bind, distort, or unwind the origin of replication. However, unwinding of DNA by T antigen was strongly inhibited by topo I when it was initiated at sites other than the origin. We demonstrate that the presence of T antigen binding sites in DNA interfere with inhibition of unwinding by topo I. These results indicate that topo I may increase the specificity of unwinding by inhibiting the reaction at non-origin sites. Fragments of T antigen that bind to topo I abrogate topo I's inhibition of non-origin-dependent unwinding, indicating that topo I inhibits unwinding through a direct interaction with T antigen. We propose a model whereby T antigen and topo I function together at the origin to specifically unwind it and initiate DNA replication.

When simian virus 40 (SV40) large T antigen binds to the virus origin of replication, it forms a double hexamer that functions as a helicase to unwind the DNA bidirectionally. We demonstrate in this report that T antigen can unwind and release an origin DNA single strand of less than full length in the presence of purified human topoisomerase I. The sites nicked by topoisomerase I in the strands released by T antigen during DNA unwinding were localized primarily to the "late" side of the origin, and the template for lagging strand synthesis was preferred significantly over the one for leading strand synthesis. Importantly, these sites were, for the most part, different from the sites nicked by topoisomerase I in the absence of T antigen. These data indicate that T antigen activates topoisomerase I nicking at discrete sites and releases these nicked strands during unwinding. We hypothesize that a single molecule of topoisomerase I can form a functional complex with a double hexamer of T antigen to simultaneously relax and unwind double-stranded origincontaining DNA.Our understanding of mammalian DNA replication has originated mostly from work on simian virus 40 (SV40). The virus DNA has a single origin of replication that has been extensively characterized. This origin is a tripartite stretch of DNA consisting of a central pentanucleotide repeat that serves as the binding sites for T antigen (the virus initiator protein) (1, 2), an early palindrome from where melting originally takes place (3, 4), and an A/T-rich track that is structurally distorted by T antigen (5-7). All three regions are required for DNA replication (1, 2, 8) and for origin unwinding (9). Neighboring sequences improve the efficiency of replication but are not absolutely required (1, 10). These auxiliary sequences are located on both sides of the origin and may facilitate the unwinding reaction (11).A great deal of effort has gone into trying to understand the composition of the initiation complex at the origin. In the presence of ATP, T antigen forms a double hexamer that completely protects the core origin from accessibility to . This double hexamer functions as an efficient helicase (16 -18) that unwinds the DNA in both directions (19). At least three cellular proteins have been shown to interact with T antigen and are believed to be recruited to the origin to form a functional initiation complex. These include DNA polymerase ␣-primase (20 -23), replication protein A (RPA) (24 -26), and topoisomerase I (27). The order in which these three proteins bind is not known; nor do we know if all proteins are present simultaneously.Recently, the work in our lab has concentrated on the interaction between T antigen and topoisomerase I. A complex between these two proteins readily forms in vitro (28), but it has been difficult to demonstrate binding in vivo.1 Nevertheless, there are a number of reasons for thinking that an interaction between T antigen and topoisomerase I has functional significance during SV40 DNA replication. First, topoisomerase I i...

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