Plasmids are important causes of drug resistance in bacteria, and serve as useful models in investigating DNA replication and its control. We have evidence that the recD gene of E. coli, a component of the recBCD gene complex, is involved in preventing rolling circle replication from occurring within the cell. When recD is inactivated, we would propose that rolling circle replication occurs from plasmids.. this replication would require the remaining RecBC nuclease to be functional; it would require a functional plasmid origin of replication; and it would generate linear plasmid multimers which can be resolved by site-specific recombination to produce covalently closed circular plasmid DNA. The linear multimers generated would also serve to disrupt normal replication control, possibly by causing overproduction of an initiator or titration of a repressor of initiation. We propose to: (1) obtain a better estimate of the extent of LPM formation by utilizing a gentle isolation technique and pulsed-field electrophoresis to estimate the size of the LPM's formed; (2) determine if normal initiation of plasmid replication is essential for LPM formation by examining the synthesis of plasmid DNA in a recD host under what are normally non-permissive conditions; (3) determine if the chromosomal origin of replication can initiate rolling circle replication by examining total DNA from recD cells for a relative excess of DNA from the origin region of the chromosome. We will also develop an in vitro DNA replication system capable of generating linear plasmid multimers. Our approach will be to adapt a widely applicable cell extract that is able to support the replication of a number of plasmids, modifying it so that it contains the proper additional components that will allow it to support rolling circle replication of a Co1E1-type plasmid. This system will then be used to determine the enzymatic requirements for such replication, its sensitivity to replication control, and to generate replicative intermediates suitable for electon microscopic examination. Once developed, such a system should be useful to a variety of investigations into DNA replication control, or the relationship of recombination to DNA replication.
