Linear plastid DNA molecules with discrete ends in WT plastids

Restriction enzymes that cleave plastid DNA rarely (once or twice) have been used to map the ends of linear molecules in high MW DNA prepared in agarose plugs. When Z. mays plastid DNA was cleaved with an enzyme that cuts once, the predicted linear 140 kbp genome band was observed. In addition, discrete smaller

Fig. 4. Models for generation of linear hairpin plastid DNA molecules by a) Template strand switching at the replication fork (Ellis and Day 1986), b) Intra-strand annealing at inverted repeat sequences (Qin and Cohen 2000). A double-strand break (DSB) initiates the pathway, which involves exonuclease and fold-back of single-stranded DNA at inverted repeats. c) A bacteriophage N15-like mechanism involving DNA cleavage at two sites, fold-back and repair of ends to form hairpins and replication followed by cleavage at one site to form a linear palindrome (Rybchin and Svarchevsky 1999). Converging box arrows indicate inverted repeats.

Fig. 4. Models for generation of linear hairpin plastid DNA molecules by a) Template strand switching at the replication fork (Ellis and Day 1986), b) Intra-strand annealing at inverted repeat sequences (Qin and Cohen 2000). A double-strand break (DSB) initiates the pathway, which involves exonuclease and fold-back of single-stranded DNA at inverted repeats. c) A bacteriophage N15-like mechanism involving DNA cleavage at two sites, fold-back and repair of ends to form hairpins and replication followed by cleavage at one site to form a linear palindrome (Rybchin and Svarchevsky 1999). Converging box arrows indicate inverted repeats.

sub-genomic bands were found (Oldenburg and Bendich 2004b). These sub-genomic bands can be explained if they arise from long linear DNA molecules containing one natural end found in vivo and one site created by restriction enzyme cleavage. These natural ends in sub-genomic fragments map to the large inverted repeats of Z. mays plastid DNA. A similar but more detailed analysis on high MW N. tabacum plastid DNA identified eleven natural ends (Scharff and Koop 2006). The majority of breaks mapped to the large inverted repeats but ends were also found in the large and small single copy regions (Scharff and Koop 2006). Some of the Z. mays and N. tabacum ends map close to plastid DNA sequences promoting DNA synthesis or exhibiting features resembling D-loops or replication bubbles (see Section 4.1 below). Only one end corresponded to the proposed site for initiation of rolling circle replication (Kolodner and Tewari 1975) located at 180° from the two D-loops (Section 4; Fig. 5a) in N. tabacum (Scharff and Koop 2006). These mapped ends define the termini of linear DNA present in high MW plastid DNA complexes (Oldenburg and Bendich 2004b; Scharff and Koop 2006). The structures of the ends of these linear sections of Z. mays and N. tabacum plastid DNA are not known but their elucidation (e.g. protected or exposed, hairpin or secondary DNA structure, or simply double-strand

DNA breaks with flush or 5' or 3' protruding ends) is likely to provide information on the mechanisms underlying their formation.

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