Initiation of rolling-circle replication in pT181 plasmid: initiator protein enhances cruciform extrusion at the origin.

Plasmid pT181 DNA secondary structures have been analyzed in vitro by nuclease S1 digestion and in vivo by bromoacetaldehyde treatment. A cruciform structure occurring at the pT181 replication origin in vitro is greatly enhanced by the binding of the plasmid-encoded initiator protein RepC. In vivo a DNA secondary structure also existed in the replication origin. Its frequency of formation was correlated with efficiency of RepC utilization. These data suggest that cruciform extrusion at the origin is involved in initiation of pT181 rolling-circle replication. A neighboring DNA structure influences the conformation of the origin in vivo. IMAGES:

本研究采用S1核酸酶（nuclease S1）体外消化法与溴乙醛（bromoacetaldehyde）体内处理法，对质粒pT181的DNA二级结构进行了分析。体外实验中，位于pT181复制原点（replication origin）处的十字形结构（cruciform structure）的形成，可因质粒编码的起始蛋白RepC（initiator protein RepC）的结合而显著增强。体内实验中，该复制原点区域同样存在DNA二级结构，其形成频率与RepC的作用效率存在显著相关性。上述实验数据表明，复制原点处的十字形挤出（cruciform extrusion）过程参与了pT181滚环复制（rolling-circle replication）的起始调控。邻近的DNA二级结构可在体内影响该复制原点的构象。图像：