Figure 1.

Reconstitution of SPP1 oriL-dependent θ-type DNA replication with B. subtilis and SPP1 purified proteins. (A) Diagram of the oriL-borne plasmid DNA templates used. Plasmid pBT430 only contains the 356-bp oriL region (StyI–HindIII fragment). Plasmid pCB163 has a 2.7-kb SPP1 region cloned (from gene 37 to gene 38). In the bottom, the oriL region is enlarged. Its is composed by a series of repeated elements called AB boxes where G38P specifically binds, and the AT-rich adjacent region known as DUE. (B) Quantification of total DNA synthesis obtained after 15 min incubation using the DNA templates: pUC18 as negative control, and pBT430 and pCB163, both carrying oriL. In lane 4, DNA synthesis obtained with PstI-linearized pCB163. The values represented are the mean of three independent experiments. (C) Time course of DNA synthesis obtained using pBT430 (□) or pCB163 (○) plasmids (both carrying oriL), quantified by incorporation of [α-³²P]dATP. (D) The DNA products generated in the replication reaction performed with pCB163 as DNA template were separated on a 0.7% alkaline agarose gel to visualize leading and lagging strand synthesis. M: 3′-labeled EcoRI-digested SPP1 DNA marker.