Bjornson K P, Hsieh J, Amaratunga M, Lohman T M
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Biochemistry. 1998 Jan 20;37(3):891-9. doi: 10.1021/bi9719307.
Escherichia coli Rep helicase is a DNA motor protein that unwinds duplex DNA as a dimeric enzyme. Using fluorescence probes positioned asymmetrically within a series of single-stranded (ss) oligodeoxynucleotides, we show that ss-DNA binds with a defined polarity to Rep monomers and to individual subunits of the Rep dimer. Using fluorescence resonance energy transfer and stopped-flow techniques, we have examined the mechanism of ss-oligodeoxynucleotide binding to preformed Rep dimers in which one binding site is occupied by a single-stranded oligodeoxynucleotide, while the other site is free (P2S dimer). We show that ss-DNA binding to the P2S Rep dimer to form the doubly ligated P2S2 dimer occurs by a multistep process with the initial binding step occurring relatively rapidly with a bimolecular rate constant of k1 = approximately 2 x 10(6) M-1 s-1 [20 mM Tris (pH 7.5), 6 mM NaCl, 5 mM MgCl2, 5 mM 2-mercaptoethanol, and 10% (v/v) glycerol, 4 degrees C]. A minimal kinetic mechanism is proposed which suggests that the two strands of ss-DNA bound to the Rep homodimer are kinetically distinct even within the P2S2 Rep dimer, indicating that this dimer is functionally asymmetric. The implications of these results for the mechanisms of DNA unwinding and translocation by the functional Rep dimer are discussed.
大肠杆菌Rep解旋酶是一种DNA马达蛋白，作为二聚体酶可解开双链DNA。我们使用荧光探针不对称地定位在一系列单链（ss）寡脱氧核苷酸内，结果表明ss-DNA以特定的极性与Rep单体以及Rep二聚体的各个亚基结合。利用荧光共振能量转移和停流技术，我们研究了ss-寡脱氧核苷酸与预先形成的Rep二聚体的结合机制，其中一个结合位点被单链寡脱氧核苷酸占据，而另一个位点是自由的（P2S二聚体）。我们发现，ss-DNA与P2S Rep二聚体结合形成双连接的P2S2二聚体是一个多步骤过程，初始结合步骤相对较快，双分子速率常数k1约为2×10⁶ M⁻¹ s⁻¹ [20 mM Tris（pH 7.5）、6 mM NaCl、5 mM MgCl₂、5 mM 2-巯基乙醇和10%（v/v）甘油，4℃]。我们提出了一个最小动力学机制，该机制表明即使在P2S2 Rep二聚体内，与Rep同二聚体结合的ss-DNA的两条链在动力学上也是不同的，这表明该二聚体在功能上是不对称的。我们还讨论了这些结果对功能性Rep二聚体进行DNA解旋和转运机制的影响。
