Human Topoisomerase I Promotes Initiation of Simian Virus 40 DNA Replication In Vitro

Abstract

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...

A Novel Nuclear Localization Signal in Human DNA Topoisomerase I

“…Thus, topo I plays an important role in different aspects of DNA metabolism such as DNA replication, DNA recombination, transcription, and, possibly, DNA repair (2)(3)(4). In addition to its catalytic activity on DNA, the enzyme has been shown to have other activities functioning as a ribonuclease and a kinase (5-7); its kinase activity has been shown to phosphorylate RNA splicing factors (5).…”

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

“…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.…”

Initiation of DNA Replication from Closed Circular DNA

“…Although RPA and pol/prim are absolutely essential for DNA synthesis (Simmons 2000), some DNA synthesis can take place in vitro in the absence of topo I (Simmons, Trowbridge et al 1998). However, topo I stimulates replication by up to 10-folds (Trowbridge, Roy et al 1999). This observation holds for assays performed with crude cell extracts depleted of topo I as well as for in vitro assays with purified proteins.…”

“…This observation holds for assays performed with crude cell extracts depleted of topo I as well as for in vitro assays with purified proteins. Topo I is needed for the efficient formation of completed form I DNA molecules as well as for the formation of replicative intermediates (Trowbridge, Roy et al 1999). The cellular enzyme is needed from the beginning of DNA synthesis and is most likely a component of the replication initiation machine (Halmer, Vestner et al 1998;Trowbridge, Roy et al 1999).…”

“…Topo I is needed for the efficient formation of completed form I DNA molecules as well as for the formation of replicative intermediates (Trowbridge, Roy et al 1999). The cellular enzyme is needed from the beginning of DNA synthesis and is most likely a component of the replication initiation machine (Halmer, Vestner et al 1998;Trowbridge, Roy et al 1999). This was demonstrated in a number of ways, but the most convincing was that reactions started with catalytically inactive mutants of topo I could not be rescued by the later addition of WT topo I suggesting that once topo I becomes incorporated into the initiation complex, it cannot be substituted by soluble enzyme.…”