Research on plastid DNA replication has been heavily influenced by the 'replicon model' put forward by Jacob, Cuzin, and Brenner (Jacob et al. 1963). The model proposes a specific DNA element that is recognised by an initiator protein. If plastid DNA replication conforms to the model it would predict initiation of replication at specific sites in plastid DNA. Replication of bacterial genomes and plas-mids in bacteria and S. cerevisiae conform to the 'replicon model' and involve origins of replication recognised by specific origin recognition proteins (Gilbert 2004). A variety of methods have been used to try and localise origins of replication in plastid genomes. Early electron microscopy (EM) studies on DNA isolated from P. sativum and Z. mays chloroplasts identified structures resembling D-loops and rolling circles (Kolodner and Tewari 1975). These are well known DNA replication intermediates and provided early models for plastid DNA replication. Unidirectional replication from an origin of replication creates a displacement loop (D-loop), comprised of double stranded DNA and a displaced single stranded DNA loop. Two D-loops spaced 7 kbp apart were found in monomer circles of P. sativum plastid DNA and gave rise to the dual D-loop model (Fig. 5a) for initiation of chloroplast DNA replication (Kolodner and Tewari 1975). Convergent replication forks from the two D-loops pass each other and a bidirectional replication bubble is formed once the forks pass the starting points of replication. The complete genome is replicated by the replication forks continuing round the circle in opposite directions, with discontinuous replication on the lagging strands, until they meet at 180o from the origin of D-loop synthesis (Fig. 5a). For rolling circle replication, a replication fork displaces the lagging strand at a nick and continues round the circle (Fig. 5b). Rolling circle replication enables multiple tandem head-to-tail copies of plastid DNA to be made from a single round of replication initiation. The rolling circles appeared to be initiated at the terminus of bidirectional replication (Kolodner and Tewari 1975). This early EM study stimulated research to locate D-loops on sequence maps of plastid DNA.
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