Abstract
DNA replication terminus (ter)-binding protein (TBP) in Escherichia coli binds specifically to the terminus (ter) site, and the resulting complex severely blocks DNA replication in an unique orientation by inhibiting the action of helicases. To generalize the intrinsic nature of the orientated ter-TBP complex against various helicases, we tested the potential of the complex to inhibit the action of three helicases, DNA helicase I, simian virus 40 (SV40) large tumor (T) antigen, and helicase B, derived from F plasmid, SV40, and mouse FM3A cell, respectively. The complex impeded the unwinding activities of all tested helicases in a specific orientation, with the same polarity observed in case of blockage of a replication fork, and, as a result, there was a block of SV40 DNA replication in both crude and purified enzyme systems in vitro. As the specificity in polarity of inhibition extends to heterologous systems, there may be common structure/mechanism features in helicases.
Highlights
From the $Laboratoryof Gene Expression and Regulation, National Institute for Basic Biology, 38 Nishigonaka, Myodaiji-cho, Okuzaki 444, Japan, the
From the in vitro reconstructed oriC plasmid replication haicaanonngetmldatrmiiicgipnnaoelsussneteixs,cIeFv,tasnMoainamrid3tinuoAiharuhnieshbeoleifitlctihvatchcsieaBreeeosu,lrealdsis,ce,etnw4rrito0ieeavnstetpeedoedsf(cttfeetStrhidrVvo-rt4emTehl0eBlyea)h.PFrpeTgclooeiphctmleeactaunopssmetmmliesaoSx,pildrDVle,N4ox(Tf0A,)theswndayeenascpsiteeensnfmsodavueroinntyfrdtofEootr.rhnectachobotleoinnttwsheotrritmtuphhcrietontehtatdeeetiirnsotyesnoserttqrheseueemaerqcnuticientoehnntcaa.hennTedaTensrBTamdmBPpinePua,armtipcifopaiaennendanrroeeTscraBccatuotPsiro,biinnnie,t impeded the unwindinagctivities of all tested helicases uiuo (Lee et al.,1989)
DNA helicase, an enzyme with the potential to unwind the served in case of blockage of a replication fork, and, double-stranded DNA to become a single strand, is thought as a result, therewas a block ofSV40DNA replication to head the DNA replication machinery, and DNA synthesis in both crude and purified enzyme systeinmvsitro
Summary
Masumi HidakaSB,Takehiko KobayashiS, Yukio Ishimill, MasaySuekkiiII,Takemi Enomotoll, Mahmoud Abdel-Monem**, and Takashi HoriuchiS. (TBP)in Escherichiacoli binds to thteer- encoded by the tuu(tus) gene, which binds to the minus (ter) site, and the resulting complex severely ter sequence UvrD proteins, were all inhibited by the complex only in one direction, with the same polarity as observed for blockage of a replication fork in uiuo. A DNA replication ter- To examine the generality of theter-TBP complex to mination site, which severely arrests the DNA replication inhibit various types of helicases, we applied the TerB-TBP fork, is present on the plasmid R6K (Bastia et al.,1981; complex in E. coli to thesimian virus 40 (SV40) in vitro DNA. One factor is the ter sequence of -22 opposite of what our evidence suggests. bp,’ a pair omr ore of which is arranged in an inverted position on the DNA molecule (Hidaka et al, 1988; Hill et al, 1988)
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