Heat shock protein-mediated disassembly of nucleoprotein structures is required for the initiation of bacteriophage lambda DNA replication.
Heat shock protein-mediated disassembly of nucleoprotein structures is required for the initiation of bacteriophage lambda DNA replication.
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发表时间：
1989-06
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通讯作者：
C. Alfano;R. McMacken
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作者：
C. Alfano;R. McMacken
三种大肠杆菌热休克蛋白，DnaJ，DnaK和GrpE，是体内噬菌体λ染色体复制所必需的。我们表明，GrpE热休克蛋白是不需要启动的λ DNA复制在体外时，DnaK的浓度是足够高的。然而，当DnaK浓度降低到亚饱和水平时，GrpE确实大大增强了DnaK在引发过程中的作用。我们在随附的文章中演示（Alfano，C.和McMacken，R.（1989）J.Biol.Chem.264，10699-10708; Dodson，M.，McMacken，R.，和Echols，H.（1989）J.Biol.Chem.264，10719-10725），DnaJ和DnaK与在λ复制起点（ori λ）处组装的预引发核蛋白结构结合。DnaJ和DnaK的结合完成了ori λ起始复合物的有序组装，ori λ起始复合物还包含λ O和P起始物以及E. coliDnaB解旋酶。随着ATP的加入，DnaJ和DnaK热休克蛋白介导起始复合物的部分分解，并且P和DnaJ蛋白大部分从模板中去除。同时，在超螺旋ori λ质粒模板上，DnaB的固有解旋酶活性被激活，DnaB启动DNA双链体的局部解旋，从而制备用于引发和DNA链延伸的模板。我们从我们的结果推断，DnaK和DnaJ在正常E.大肠杆菌代谢，促进ATP依赖的蛋白质解折叠和分解反应。我们还提供了证据表明，无论是λ O和P引发剂，也没有E。coli DnaJ和DnaK热休克蛋白在体外λ复制叉的增殖中起直接作用。
Three Escherichia coli heat shock proteins, DnaJ, DnaK, and GrpE, are required for replication of the bacteriophage lambda chromosome in vivo. We show that the GrpE heat shock protein is not required for initiation of lambda DNA replication in vitro when the concentration of DnaK is sufficiently high. GrpE does, however, greatly potentiate the action of DnaK in the initiation process when the DnaK concentration is reduced to a subsaturating level. We demonstrate in the accompanying articles (Alfano, C. and McMacken, R. (1989) J. Biol. Chem. 264, 10699-10708; Dodson, M., McMacken, R., and Echols, H. (1989) J. Biol. Chem. 264, 10719-10725) that DnaJ and DnaK bind to prepriming nucleoprotein structures that are assembled at the lambda replication origin (ori lambda). Binding of DnaJ and DnaK completes the ordered assembly of an ori lambda initiation complex that also contains the lambda O and P initiators and the E. coli DnaB helicase. With the addition of ATP, the DnaJ and DnaK heat shock proteins mediate the partial disassembly of the initiation complex, and the P and DnaJ proteins are largely removed from the template. Concomitantly, on supercoiled ori lambda plasmid templates, the intrinsic helicase activity of DnaB is activated and DnaB initiates localized unwinding of the DNA duplex, thereby preparing the template for priming and DNA chain elongation. We infer from our results that DnaK and DnaJ function in normal E. coli metabolism to promote ATP-dependent protein unfolding and disassembly reactions. We also provide evidence that neither the lambda O and P initiators nor the E. coli DnaJ and DnaK heat shock proteins play a direct role in the propagation of lambda replication forks in vitro.