Analysis of the tobacco chloroplast DNA replication origin (oriB) downstream of the 23 S rRNA gene.

We have mapped the origin of DNA replication (oriB) downstream of the 23 S rRNA gene in each copy of the inverted repeat (IR) of tobacco chloroplast DNA between positions 130,502 and 131,924 (IR(A)) by a combination of approaches. In vivo chloroplast DNA replication intermediates were examined by two-dimensional agarose gel electrophoresis. Extended arc patterns suggestive of replication intermediates containing extended single-stranded regions were observed with the 4.29 kb SspI fragment and an overlapping EcoRI fragment from one end of the inverted repeat, while only simple Y patterns were observed with a 3.92 kb BamHI-KpnI fragment internal to the SspI fragment. Other restriction fragments of tobacco chloroplast DNA besides those at the oriA region also generated only simple Y patterns in two-dimensional agarose gels. Several chloroplast DNA clones from this region were tested for their ability to support in vitro DNA replication using a partially purified chloroplast protein fraction. Templates with a deletion of 154 bp from the SspI to the BamHI sites near the end of the inverted repeat resulted in a considerable loss of in vitro DNA replication activity. These results support the presence of a replication origin at the end of the inverted repeat. The 5' end of nascent DNA from the replication displacement loop was identified at position 130,697 for IR(A) (111,832 for IR(B)) by primer extension. A single major product insensitive to alkali and RNase treatment was observed and mapped to the base of a stem-loop structure which contains one of two neighboring BamHI sites near the end of each inverted repeat. This provides the first precise determination of the start site of DNA synthesis from oriB. Adjacent DNA fragments containing the stem-loop structure and the 5' region exhibit sequence-specific gel mobility shift activity when incubated with the replication protein fraction, suggesting the presence of multiple binding sites.