Differential inhibition of the DNA translocation and DNA unwinding activities of DNA helicases by the Escherichia coli Tus protein.
Binding of the Escherichia coli Tus protein to its cognate nonpalindromic binding site on duplex DNA (a Ter sequence) is sufficient to arrest the progression of replication forks in a Ter orientation-dependent manner in vivo and in vitro. In order to probe the molecular mechanism of this inhibition, we have used a strand displacement assay to investigate the effect of Tus on the DNA helicase activities of DnaB, PriA, UvrD (helicase II), and the phi X-type primosome. When the substrate was a short oligomer hybridized to a circular single-stranded DNA, strand displacement by DnaB, PriA, and the primosome (in both directions), but not UvrD, was blocked by Tus in a polar fashion. However, no inhibition of either DnaB or UvrD was observed when the substrate carried an elongated duplex region. With this elongated substrate, PriA helicase activity was only inhibited partially (by 50%). On the other hand, both the 5'----3' and 3'----5' helicase activities of the primosome were inhibited almost completely by Tus with the elongated substrate. These results suggest that while Tus can inhibit the translocation of some proteins along single-stranded DNA in a polar fashion, this generalized effect is insufficient for the inhibition of bona fide DNA helicase activity.
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Program in Molecular Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York.
Marians
K J
KJ
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001483:Base Sequence; D004265:DNA Helicases / Q000037:antagonists & inhibitors*; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053488:DnaB Helicases; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D051716:Replication Protein A; D013379:Substrate Specificity; D013728:Terminator Regions, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D051716:Replication Protein A; C061910:tus protein, Bacteria; C489236:tus protein, E coli; D004265:DNA Helicases; D053488:DnaB Helicases