A hybrid bacterial replication origin

Abstract

We constructed a hybrid replication origin that consists of the main part of oriC from Escherichia coli, the DnaA box region and the AT-rich region from Bacillus subtilis oriC. The AT-rich region could be unwound by E. coli DnaA protein, and the DnaB helicase was loaded into the single-stranded bubble. The results show that species specificity, i.e. which DnaA protein can do the unwinding, resides within the DnaA box region of oriC.

Fig. 1. Alignment of AT-rich regions from E. coli, B. subtilis and pOCBS oriC. The regions are aligned using the adjacent DnaA box (shaded). AT cluster, 13mers and 27mers are shaded. Potential ATP–DnaA boxes are underlined. KMnO₄-reactive pyrimidines are indicated by arrows. Escherichia coli sequences from positions 10 to 93 (DDBJ/EMBL/GenBank accession No. K00826) and B. subtilis sequences positions 2706 to 2623 (DDBJ/EMBL/GenBank accession No. X02369) are shown. Colons are at every 10th position.

Fig. 2. KMnO₄ footprinting of pOCBS and pOC180 oriC. The footprinting reaction was done with 1 µg ccc plasmid DNA and 0, 250, 500 and 750 ng DnaA as described in the Methods. Sequencing reactions of pOCBS were used as standards and obtained using the same labelled primer A as for the KMnO₄ footprints. The bar indicates the 28 bp region that is unwound in E. coli and B. subtilis (Krause and Messer, 1999) (also see Figure 1).

Fig. 3. FI* helicase loading assay. The FI* assay was done as described in the Methods.