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Unique features of the mitochondrial rolling circle-plasmid mp1 from the higher plant Chenopodium album (L.)
S Backert1, K Meissner, T Börner
1Institut für Biologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, D-10115 Berlin, Germany.
Abstract:
We analyzed the structure and replication of the mitochondrial (mt) circular DNA plasmid mp1 (1309 bp) from the higher plant Chenopodium album(L.). Two dimensional gel electrophoresis (2DE) revealed the existence of oligomers of up to a decamer in addition to the prevailing monomeric form. The migration behavior of cut replication intermediates during 2DE was consistent with a rolling circle (RC) type of replication. We detected entirely single-stranded (ss) plasmid copies hybridizing only with one of the two DNA strands. This result indicates the occurence of an asymmetric RC replication mechanism. mp1 has, with respect to its replication, some unique features compared with bacterial RC plasmids. We identified and localized a strand-specific nicking site (origin of RC replication) on the plasmid by primer extension studies. Nicks in the plasmid were found to occur at any one of six nucleotides (TAAG/GG) around position 735 of the leading strand. This sequence shows no homology to origin motifs from known bacterial RC replicons. mp1 is the first described RC plasmid in a higher plant.
Insights
This study reveals the unique rolling circle replication mechanism of the mp1 plasmid in the higher plant Chenopodium album. It details the asymmetric replication and identifies a novel origin of replication not found in bacterial plasmids.
Area of Science:
- Plant molecular biology
- Mitochondrial DNA replication
- Plasmid biology
Background:
- Mitochondrial DNA (mtDNA) plasmids are found in various organisms, but their replication mechanisms in higher plants remain poorly understood.
- The mp1 plasmid from Chenopodium album is a circular DNA molecule residing in plant mitochondria.
Purpose of the Study:
- To elucidate the replication mechanism of the mp1 mitochondrial plasmid in Chenopodium album.
- To characterize the structural features and replication intermediates of mp1.
- To identify the origin of replication and compare it with known systems.
Main Methods:
- Two-dimensional gel electrophoresis (2DE) to analyze DNA structures and replication intermediates.
- Hybridization studies to detect single-stranded DNA.
- Primer extension assays to identify and localize the nicking site.
Main Results:
- mp1 exists as monomers and oligomers (up to decamers) in mitochondria.
- Replication intermediates are consistent with a rolling circle (RC) mechanism.
- Asymmetric replication leads to entirely single-stranded DNA intermediates.
- A novel, strand-specific nicking site (origin of RC replication) was identified at position 735 on the leading strand, showing no homology to bacterial RC origins.
- mp1 represents the first described RC plasmid in a higher plant.
Conclusions:
- The mp1 plasmid utilizes an asymmetric rolling circle replication mechanism in higher plant mitochondria.
- This mechanism possesses unique features distinct from bacterial RC plasmids.
- The identified nicking site represents a novel origin of replication for plant mitochondrial plasmids.