Hiasa H, Marians K J
Program in Molecular Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York.
J Biol Chem. 1992 Jun 5;267(16):11379-85.
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.
大肠杆菌Tus蛋白与其在双链DNA上同源的非回文结合位点（Ter序列）的结合足以在体内和体外以Ter方向依赖的方式阻止复制叉的前进。为了探究这种抑制作用的分子机制，我们使用链置换分析来研究Tus对DnaB、PriA、UvrD（解旋酶II）和phi X型引发体的DNA解旋酶活性的影响。当底物是与环状单链DNA杂交的短寡聚物时，DnaB、PriA和引发体（双向）的链置换，但不是UvrD的链置换，会被Tus以极性方式阻断。然而，当底物带有延长的双链区域时，未观察到对DnaB或UvrD的抑制作用。对于这种延长的底物，PriA解旋酶活性仅被部分抑制（50%）。另一方面，对于延长的底物，引发体的5'→3'和3'→5'解旋酶活性几乎完全被Tus抑制。这些结果表明，虽然Tus可以以极性方式抑制某些蛋白质沿单链DNA的易位，但这种普遍效应不足以抑制真正的DNA解旋酶活性。
