Kubota Y, Arai K, Masai H
Department of Molecular and Developmental Biology, University of Tokyo, Japan.
Gene. 1993 Apr 15;126(1):9-16. doi: 10.1016/0378-1119(93)90584-p.
Bacterial replicons often contain single-strand initiation sequences (ssi) such as a G site (a sequence recognized by a dnaG-encoded primase for the synthesis of primer RNA) and a primosome assembly site (pas) near the DNA replication origin (ori). The R1 plasmid contains a G site downstream from oriR, which serves for the priming of the leading-strand synthesis of this plasmid. On the other hand, the F, R6K and Rts1 plasmids carry pas at similar locations relative to the respective ori. In order to assess the functional significance of these pas, R1 plasmid derivatives carrying an n'-pas (phi X174-type pas) in place of the G site were constructed and their replication properties were examined in vitro. Deletion of the G site in the R1 plasmid resulted in a nearly 80% reduction of total DNA synthesis in vitro, which was recovered to the wild-type (wt) level by inserting the G4 complementary ori. Furthermore, insertion of an n'-pas on the leading-strand template restored the in vitro replicative activity to a level 70% of wt. This recovery was dependent on the assembly of the phi X174-type primosome, which efficiently primed leading-strand synthesis and moved toward the oriR. However, the R1 plasmid derivative containing the n'-pas replicated unidirectionally in vitro, probably due to the anti-helicase activity of the RepA protein bound to oriR, which was shown by helicase assays using partial heteroduplexes as substrates.(ABSTRACT TRUNCATED AT 250 WORDS)
细菌复制子通常包含单链起始序列（ssi），如G位点（一种由dnaG编码的引发酶识别的用于合成引物RNA的序列）和靠近DNA复制起点（ori）的引发体组装位点（pas）。R1质粒在oriR下游含有一个G位点，用于该质粒前导链合成的引发。另一方面，F、R6K和Rts1质粒在相对于各自ori的相似位置携带pas。为了评估这些pas的功能意义，构建了携带n'-pas（phi X174型pas）代替G位点的R1质粒衍生物，并在体外检测了它们的复制特性。R1质粒中G位点的缺失导致体外总DNA合成减少近80%，通过插入G4互补ori可恢复到野生型（wt）水平。此外，在前导链模板上插入n'-pas可将体外复制活性恢复到wt水平的70%。这种恢复依赖于phi X174型引发体的组装，其有效地引发前导链合成并向oriR移动。然而，含有n'-pas的R1质粒衍生物在体外单向复制，这可能是由于与oriR结合的RepA蛋白的抗解旋酶活性，这通过使用部分异源双链体作为底物的解旋酶测定得以证明。（摘要截短于250字）
