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The replication of plastid minicircles involves rolling circle intermediates.

Leung SK, Wong JT - Nucleic Acids Res. (2009)

Bottom Line: Plastid genomes of peridinin-containing dinoflagellates are unique in that its genes are found on multiple circular DNA molecules known as 'minicircles' of approximately 2-3 kb in size, carrying from one to three genes.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.Accumulating data thus supports the involvement of rolling circle intermediates in the replication of the minicircles.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Hong Kong University of Science and Technology, Kowloon, Hong Kong SAR, PR China.

ABSTRACT
Plastid genomes of peridinin-containing dinoflagellates are unique in that its genes are found on multiple circular DNA molecules known as 'minicircles' of approximately 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.

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Effects of Topo II and aphidicolin on the minicircle DNA. (A) Topo II-treated whole-DNA extract from H. triquetra, and positive control of untreated whole DNA, were subjected to PFGE, and detected with 1.1-kb psbA NCR fragment. (B) Aphidicolin-treated whole-DNA extracts collected after 12- and 18-h incubation (with parallel untreated controls) were hybridized with the same probe after PFGE. DNA markers were linear DNAs from a commercial source (Invitrogen Corporation).
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Figure 3: Effects of Topo II and aphidicolin on the minicircle DNA. (A) Topo II-treated whole-DNA extract from H. triquetra, and positive control of untreated whole DNA, were subjected to PFGE, and detected with 1.1-kb psbA NCR fragment. (B) Aphidicolin-treated whole-DNA extracts collected after 12- and 18-h incubation (with parallel untreated controls) were hybridized with the same probe after PFGE. DNA markers were linear DNAs from a commercial source (Invitrogen Corporation).

Mentions: To verify whether the APBs are catenated minicircles, an in vitro Topo II assay was performed. Topo II is able to decatenate topologically linked circular DNA molecules by producing a transient double-stranded break (33). The decatenated products after resealing would be DNA in either the open circular or relaxed circular forms. Heterocapsa triquetra whole DNA was treated with Topo II and detected for minicircle DNA after PFGE. The APBs suspected to be linked minicircles should be removed due to decatenation by Topo II. In Figure 3A, however, the APBs were still detected in Topo II-treated DNA by the psbA NCR probe. This result indicates that the APBs are not catenated minicircles. Three extra bands with sizes around 2 kb were observed too, suggesting the presence of smaller minicircle DNA detected by the psbA NCR fragment.Figure 3.


The replication of plastid minicircles involves rolling circle intermediates.

Leung SK, Wong JT - Nucleic Acids Res. (2009)

Effects of Topo II and aphidicolin on the minicircle DNA. (A) Topo II-treated whole-DNA extract from H. triquetra, and positive control of untreated whole DNA, were subjected to PFGE, and detected with 1.1-kb psbA NCR fragment. (B) Aphidicolin-treated whole-DNA extracts collected after 12- and 18-h incubation (with parallel untreated controls) were hybridized with the same probe after PFGE. DNA markers were linear DNAs from a commercial source (Invitrogen Corporation).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665238&req=5

Figure 3: Effects of Topo II and aphidicolin on the minicircle DNA. (A) Topo II-treated whole-DNA extract from H. triquetra, and positive control of untreated whole DNA, were subjected to PFGE, and detected with 1.1-kb psbA NCR fragment. (B) Aphidicolin-treated whole-DNA extracts collected after 12- and 18-h incubation (with parallel untreated controls) were hybridized with the same probe after PFGE. DNA markers were linear DNAs from a commercial source (Invitrogen Corporation).
Mentions: To verify whether the APBs are catenated minicircles, an in vitro Topo II assay was performed. Topo II is able to decatenate topologically linked circular DNA molecules by producing a transient double-stranded break (33). The decatenated products after resealing would be DNA in either the open circular or relaxed circular forms. Heterocapsa triquetra whole DNA was treated with Topo II and detected for minicircle DNA after PFGE. The APBs suspected to be linked minicircles should be removed due to decatenation by Topo II. In Figure 3A, however, the APBs were still detected in Topo II-treated DNA by the psbA NCR probe. This result indicates that the APBs are not catenated minicircles. Three extra bands with sizes around 2 kb were observed too, suggesting the presence of smaller minicircle DNA detected by the psbA NCR fragment.Figure 3.

Bottom Line: Plastid genomes of peridinin-containing dinoflagellates are unique in that its genes are found on multiple circular DNA molecules known as 'minicircles' of approximately 2-3 kb in size, carrying from one to three genes.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.Accumulating data thus supports the involvement of rolling circle intermediates in the replication of the minicircles.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Hong Kong University of Science and Technology, Kowloon, Hong Kong SAR, PR China.

ABSTRACT
Plastid genomes of peridinin-containing dinoflagellates are unique in that its genes are found on multiple circular DNA molecules known as 'minicircles' of approximately 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.

Show MeSH