Jezewska M J, Rajendran S, Bujalowski W
Department of Human Biological Chemistry and Genetics and The Sealy Center for Structural Biology, The University of Texas Medical Branch at Galveston, 301 University Boulevard, Galveston, Texas 77555-1053, USA.
Biochemistry. 1998 Mar 3;37(9):3116-36. doi: 10.1021/bi972564u.
Interactions of the Escherichia coli replicative helicase DnaB protein, with DNA replication fork substrates, have been studied using rigorous fluorescence titration, fluorescence energy transfer, and analytical ultracentrifugation methods. DnaB binds the 5' single-arm fork, the 3' single-arm fork, and the two-arm fork with stoichiometries of 1, 1, and 2 DnaB hexamers per fork, independent of the length of the duplex part of the fork. Within the structurally heterogeneous binding site, the helicase accesses most of the 20 nucleotide residues of an arm. The dsDNA of the fork does not contribute to the affinity; however, it affects the positioning of the enzyme on the 5' or 3' arm. Fluorescence energy transfer experiments provide direct evidence that the DnaB helicase binds the 5' arm of the fork in a single orientation, with respect to the duplex part of the fork. The 33-kDa domains of the hexamer face the dsDNA, while the small 12-kDa domains face the 5' end of the arm. In the complex with the 3' arm, the helicase is bound in an opposite orientation when compared to the 5' arm. This is the first determination of the strict, single orientation of a helicase in the complex with a replication fork. The 3' arm accommodates a DnaB hexamer, while another hexamer is associated with the 5' arm. The complex of two DnaB hexamers bound in opposite orientations with each arm of the fork may play an important role during bidirectional replication of the E. coli DNA.
利用严格的荧光滴定、荧光能量转移和分析超速离心方法，对大肠杆菌复制解旋酶DnaB蛋白与DNA复制叉底物之间的相互作用进行了研究。DnaB与5'单臂叉、3'单臂叉和双臂叉结合，每个叉的化学计量比分别为1、1和2个DnaB六聚体，与叉的双链部分长度无关。在结构异质的结合位点内，解旋酶可接触到臂上20个核苷酸残基中的大部分。叉的双链DNA对亲和力没有贡献；然而，它会影响酶在5'或3'臂上的定位。荧光能量转移实验提供了直接证据，表明DnaB解旋酶相对于叉的双链部分以单一方向结合叉的5'臂。六聚体的33 kDa结构域面向双链DNA，而小的12 kDa结构域面向臂的5'端。与3'臂形成复合物时，与5'臂相比，解旋酶以相反的方向结合。这是首次确定解旋酶与复制叉形成复合物时严格的单一方向。3'臂容纳一个DnaB六聚体，而另一个六聚体与5'臂相关联。两个以相反方向与叉的每个臂结合的DnaB六聚体复合物可能在大肠杆菌DNA的双向复制过程中发挥重要作用。
