The dinoflagellate plastid genome is unique in that its genes are found on multiple circular DNA named as “minicircles” of 2-3 kb in size, carrying one to three genes. So far, only 14 typical plastid-located genes are discovered on minicircles. The non-coding regions (NCRs) of minicircles share a conserved core region of 250-500 bp. Genes carried in the minicircles are found in the same orientation with respect to the core region. All core regions are also AT-rich and have several inverted or direct repeats. However, no well-defined replication origin sequence can be identified in minicircles. Factors affecting minicircle DNA synthesis has not been studied....[ Read more ]

The dinoflagellate plastid genome is unique in that its genes are found on multiple circular DNA named as “minicircles” of 2-3 kb in size, carrying one to three genes. So far, only 14 typical plastid-located genes are discovered on minicircles. The non-coding regions (NCRs) of minicircles share a conserved core region of 250-500 bp. Genes carried in the minicircles are found in the same orientation with respect to the core region. All core regions are also AT-rich and have several inverted or direct repeats. However, no well-defined replication origin sequence can be identified in minicircles. Factors affecting minicircle DNA synthesis has not been studied.

In this study, the possible replication mechanism of minicircles was investigated. Southern blot analysis using NCR as probe revealed positive bands of 6-8 kb after resolving dinoflagellate whole DNA extract in a pulsed-field gel electrophresis. An in vitro topoisomerase assay of the DNA showed that the larger DNA molecules are not catenated minicircles. Exonuclease III treatment and two-dimensional agarose gel electrophoresis implicated the 6-8 kb minicircular DNA has linear components, probably minicircle replication intermediates produced during rolling circle type replication. Atomic force microscopy reveals possible rolling circle DNA too. The role of NCR in minicircle replication was examined. The NCR is able to specifically bind to dinoflagellate proteins, and to form secondary structure which likely involved in replication. The possible location of replication origin was also determined. An oscillation of minicircle DNA level was observed in a synchronized culture of Heterocapsa triquetra. Minicircle level was higher in light phase and lower in dark phase. Also, minicircle synthesis and nuclear genome replication were found apparently uncoupled after measurement on the changes of mincircle levels during an alternate light/dark regime experiment.