The replication of plastid minicircles involves rolling circle intermediates

Plastid genomes of peridinin-containing dinoflagellates are unique in that its genes are found on multiple circular DNA molecules known as ‘minicircles’ of ∼2–3 kb in size, carrying from one to three genes. The non-coding regions (NCRs) of these minicircles share a conserved core region (250–500 bp) that are AT-rich and have several inverted or direct repeats. Southern blot analysis using an NCR probe, after resolving a dinoflagellate whole DNA extract in pulsed-field gel electrophoresis (PFGE), revealed additional positive bands (APBs) of 6–8 kb in size. APBs preferentially diminished from cells treated with the DNA-replication inhibitor aphidicolin, when compared with 2–3 kb minicircles, implicating they are not large minicircles. The APBs are also exonuclease III-sensitive, implicating the presence of linear DNA. These properties and the migration pattern of the APBs in a 2D-gel electrophoresis were in agreement with a rolling circle type of replication, rather than the bubble-forming type. Atomic force microscopy of 6–8 kb DNA separated by PFGE revealed DNA intermediates with rolling circle shapes. Accumulating data thus supports the involvement of rolling circle intermediates in the replication of the minicircles.