Abeles A L
J Biol Chem. 1986 Mar 15;261(8):3548-55.
The minimal P1 replicon encompasses an open reading frame for the essential replication protein, RepA, bracketed by two sets of multiple 19-base pair repeated sequences, incA and incC. This study focused on the interaction of RepA with the incC and incA repeated sequences because earlier studies suggested that incA might control P1 copy number by titrating limiting amounts of RepA and because the incC repeats, which are part of the origin of replication, contain the promoter for repA. RepA is essential for origin function, autoregulates its own synthesis from the promoter, and, when overproduced, blocks origin function. In this study, RepA was overproduced from an expression vector and purified to 90% homogeneity. The binding of RepA to the DNA encompassing repeat sequences was assayed by monitoring the mobility of protein-DNA complexes on polyacrylamide gels. Distinct species of retarded bands were seen with the maximum number of bands corresponding to the number of repeats present in the target fragment. No evidence was found for RepA binding to fragments not containing the repeats. This suggests that the specific binding of RepA to the repeats may be involved in each of the diverse activities of RepA.
最小的P1复制子包含一个编码必需复制蛋白RepA的开放阅读框，其两侧是两组19个碱基对的多重重复序列，即incA和incC。本研究聚焦于RepA与incC和incA重复序列的相互作用，原因如下：早期研究表明，incA可能通过滴定有限量的RepA来控制P1拷贝数；而且作为复制起点一部分的incC重复序列包含repA的启动子。RepA对于起点功能至关重要，它能从启动子自动调节自身的合成，并且在过量表达时会阻断起点功能。在本研究中，RepA从表达载体中过量表达并纯化至90%的纯度。通过监测蛋白质 - DNA复合物在聚丙烯酰胺凝胶上的迁移率来检测RepA与包含重复序列的DNA的结合情况。观察到不同种类的滞后条带，条带的最大数量与目标片段中重复序列的数量相对应。未发现RepA与不含重复序列的片段结合的证据。这表明RepA与重复序列的特异性结合可能参与了RepA的各种不同活性。
