DNA recombination in plastids

Plastid fusion and DNA recombination between different plastid types are rare in flowering plants. Rapid segregation is observed when two plastid types with different genomes are forced into the same cell by protoplast fusion (Morgan and Maliga 1987). In C. reinhardtii, recombination between parental plastid genomes in exceptional zygotes is well established (Gillham 1974). The development of plastid transformation has demonstrated an active homologous DNA recombination pathway in C. reinhardtii (Boynton et al. 1988) and flowering plant plastids (Svab et al. 1990). The rarity of plastid fusion in angiosperms probably explains the lack of DNA recombination between "parent" plastid genomes in protoplast fusion experiments. In one successful protoplast fusion experiment a single plant with a recombinant plastid genome resulting from at least six crossover events between parental genomes was isolated (Medgyesy et al. 1985).

Most characterised plastid genomes contain a large inverted repeat sequence. Recombination between the large inverted repeat sequences (flip-flop recombination) is responsible for the two isomers of plastid DNA, which differ with respect to the orientation of the single copy regions (Palmer 1983). Flip-flop recombination giving rise to the two isomers can take place between circular (Fig. 7a) or linear DNA substrates (Fig. 7b). The head-to-head circular dimers (Fig. 3a) in L. sativa and S. oleracea plastids observed by Kolodner and Tewari (1979) were explained by intermolecular recombination between opposite large inverted repeats in circular DNA substrates (Fig. 8a). These head-to-head dimers are comprised of an inverted sequence representing ~90% of the unit genome size separated by small spacer loops comprised of the small single copy sequences. P. sativum plastid DNA lacks a large inverted repeat providing an explanation for the lack of head-to-head dimers in plastids from this species (Kolodner and Tewari 1979). Head-to-head inverted sequences representing ~90% of the unit genome length will also be produced by recombination events between large inverted repeat sequences involving linear DNA substrate (Fig. 8b). Homologous recombination that is not limited to specific sequences appears to be responsible for generating these isomers. Intermolecular recombination between inverted repeats in long chain multimers of plastid DNA would be expected to place at any point and would give rise to a large number of isomers. Intramolecular recombination events between tandemly repeated copies of the unit genome in linear multimers will give

Isomer 1 Isomer 2
Linear Dna Recombination

Isomer 2

Fig. 7. Intramolecular flip-flop recombination between large inverted repeat sequences in a) Circular, and b) Linear DNA molecules. The large inverted repeat sequences are shown as converging grey and white box arrows. Note that the linear product in b contains one of the head-to-head inverted sequences high-lighted in Fig. 8b.

Isomer 2

Fig. 7. Intramolecular flip-flop recombination between large inverted repeat sequences in a) Circular, and b) Linear DNA molecules. The large inverted repeat sequences are shown as converging grey and white box arrows. Note that the linear product in b contains one of the head-to-head inverted sequences high-lighted in Fig. 8b.

rise to circular DNA molecules. Oldenburg and Bendich (2004b) have pointed out that recombination-dependent DNA replication primed by an end within the large inverted repeat will also result in head-to-head inverted sequences and flipping of single copy regions. This can be visualised by looking at Figure 8b where the products of reciprocal recombination can also be obtained by strand-invasion by the top molecule on the bottom template at the crossover site followed by D-loop replication, resolution, and replication fork movement (see Fig. 5d) to the end of the template molecule.

Figure 9 shows a recombination event between large inverted repeats following replication of one copy of the repeat (Futcher 1986). This switches the direction of the replication fork allowing many identical head-to-tail copies of the unit genome to be made in a multimeric circle from a single template without re-initiation of DNA replication. The absence of a large inverted repeat in some plastid genomes (Palmer and Thompson 1982) would suggest that this double rolling circle mechanism is not essential for amplification of plastid DNA. A linear multimeric chain replicated from a circular template will be formed if the lagging strand is not replicated following recombination between the duplicated and unreplicated copies of the large inverted repeats (Ellis and Day 1985).

Inverted Repeat

Fig. 8. Intermolecular recombination between opposite large inverted repeat sequences gives rise to plastid genomes orientated head-to-head. a) Circular head-to-head and b) Linear head-to-head DNA molecules. Head-to-head inverted sequences are shown as dotted arrowed lines. Length is expressed as a percentage of the unit genome size.

Fig. 8. Intermolecular recombination between opposite large inverted repeat sequences gives rise to plastid genomes orientated head-to-head. a) Circular head-to-head and b) Linear head-to-head DNA molecules. Head-to-head inverted sequences are shown as dotted arrowed lines. Length is expressed as a percentage of the unit genome size.

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