The Activity of Topoisomerase I Is Modulated by Large T Antigen during Unwinding of the SV40 Origin

Abstract

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...

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

“…Topo I has no effect if it is introduced during the elongation stage (57). Also, topo I nicks origin DNA at specific and unique sites during T-antigen-mediated DNA unwinding, indicating that the interaction between T antigen and topo I is functionally significant (51). Furthermore, topo I enhances the fidelity of origin unwinding by T antigen (52).…”

“…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.…”

Host-Specific Replication of BK Virus DNA in Mouse Cell Extracts Is Independently Controlled by DNA Polymerase α-Primase and Inhibitory Activities

“…The viral core origin is sufficient to constitute a functional replication origin, but the presence of auxiliary domains increases its activity 5-to 100-fold in vivo (16,30). After the viral TAg unwinds the core origin and its flanking sequences, replication protein A (RPA), the main eukaryotic ssDNA-binding protein, covers the resulting stretches of ssDNA, whereas topoisomerase I releases the resulting torsional stress and enhances initiation of DNA replication (5,7,43). Then, DNA polymerase ␣-primase (Polprim) is loaded onto this TAg-RPA-topoisomerase 1-DNA complex, yielding a functional initiation complex.…”

DNA replication and transcription direct a DNA strand bias in the process of targeted gene repair in mammalian cells

“…Replication proceeds bidirectionally, using both leading-and lagging-strand modes of synthesis. The SV40 system was tailored for our experiments by creating four expression vectors (pSK128-eGFP-1, pSK128-eGFP-2, pSK128-eGFP-3 and pSK128-eGFP-4) derived from pSK128, a Bluescript plasmid containing an SV40 origin of replication (Simmons et al, 1998). A mutant enhanced green fluorescent protein (eGFP) gene was ligated into the pSK128 vector in both orientations and on different sides of the SV40 origin of replication so that the effect of either replication polarity and/or transcriptional status on gene repair could be monitored.…”