# Recollections of a [PERSON] Disciple
## 3 Searching for Cell Cycle-Specificity in Plasmid Systems  
With the clear cell cycle-specific replication pattern observed for minichromosomes, [PERSON] and I turned our attention to other extrachromosomal replicons in _E. coli_. At the time, plasmid systems were commonly used as surrogates to study chromosome replication, and we were intrigued by the possibility that some plasmids would behave similarly to minichromosomes, particularly the low copy types such as F factors and the many plasmids whose replication origins interact with the chromosomal initiator protein DnaA; some examples are P1 [12], pSC101 [13], R1 [14], and mini F [15]. It seemed obvious that we should use minichromosomes as an internal control for cycle-specific replication, and our studies at the time uniquely included multiple compatible replicons co-inhibiting the same _E. coli_ cell. The baby machine procedure to measure minichromosome replication during the cell cycle could be used unaltered, as long as we were careful to use plasmids of sizes that could be resolved from one another on agarose gels. While our efforts were limited to only the most prominent model systems (F, ColE1, pBR322, pSC101, and R1 derivatives), we were unable to identify any plasmid types that showed cell cycle-specific periodicity similar to minichromosomes (for example, see Figure 5, and [16]. Our F plasmid replication data from baby machine experiments was later analyzed using stochastics to reveal that the replication rate function increased monotonically over the cell cycle, with a rapid increase near cell division [17]. The resulting model is consistent with a replication control mechanism that is designed to force most plasmids to replicate before cells undergo division. Extending this model to the case of cell cycle-dependent replication requires additional, as yet unspecified control elements. Later experiments were extended to NR1 and P1 replicons [18]. OriP1 was able to initiate replication at all stages of the cell cycle with a slight periodicity observed in slower growing cells and NR1 plasmid replication was random during the cell cycle.  
Figure 5: Cell cycle replication of various plasmids. (**A**). Fluorograph of radioactive plasmid DNA in newborn cells from the effluent of a membrane filter-bound culture of _E. coli_ B/rF26 containing pSG21 mini-F, pBR322, and pAL70 simultaneously. Cells were grown, pulse-labeled, and prepared as in Figure 3. In this experiment, all lanes contained lysate from the same number of newborn cells. (**B**). Radioactive plasmid DNA in newborn cells from a membrane filter-bound culture of _E. coli_ B/r F26 containing F’ lac, pSC101, pAL49, and pBR322 simultaneously. Cells growing exponentially were pulse labeled and treated as described in Figure 3. Modified from [16].  
Some controversy about plasmid replication remains unresolved, since later studies from [PERSON]'s lab were consistent with cell cycle specific replication for R6K, P1, F, and mini-F [19; 20; 21; 22]. It is not clear why their results contrasted so dramatically with ours. Since all experiments were performed using baby machine selection, any differences in replication patterns must be due to differences in the _E. coli_ strains, the post-processing of samples, and/or the assay of radiolabeled DNA.  
We were intrigued to find that plasmids whose replication origins were known to interact with DnaA did not use this protein to couple their replication to the cell cycle. For these plasmids, DnaA availability might serve as a monitor of host metabolic activity, acting as either an on or off switch for plasmid replication depending on the plasmid type; for example [23; 24]. This function would depend on the free availability of DnaA and not necessarily the ATP-bound state of the initiator. Our inability to identify cell cycle-specific plasmid replicons also raised the question of whether cell-cycle-specific or chromosome-coupled replication is ever beneficial for plasmids. Insights may come from bacteria containing two heterologous chromosomes (see Discussion).