## 3 Discussion
Whereas, the mechanism for initiation of ColE1 [43] is similar to restart (see [35]), and likely requires loading DnaB for each initiation event, which breaks down with P-DnaB sequestration. Just how PR RifR mutants suppress ColE1 curing by P, and seemingly overcome P-DnaB sequestration remain open questions. The NH\({}_{2}\)-terminal portion of P was suggested to bind O and its C-terminal domain to interact with host proteins [86; 87]. The NH\({}_{2}\)-terminal domain of DnaC is involved in binding to DnaB. Both P and DnaC share a stretch of amino acids with high homology at their NH\({}_{2}\) termini [88]. The in-frame \(\Delta\)76 deletion within codons 9-86 at the NH\({}_{2}\)-terminal end of P suppressed: (a) the curing of ColE1 plasmids; (b) P-Iethality at 37-39 \({}^{\circ}\)C [35]; and (c) prevented a P-dependent increase in RifR mutations or mutagenic effect. These observations suggest that the NH\({}_{2}\) end of P can compete for the DnaCbinding site on DnaB, and that P binding at this site can account for P\({}^{\text{S}}\) phenotype: its cellular toxicity, ColE1 curing, and mutator activity. The cell construct, Figure 1A, possessed a mutagenic activity that was linked to the expression of \(P\) from the \(\lambda\) fragment within the chromosome. Independently, cells that included a plasmid where \(P\) was the only inducible gene product exhibited a mutator phenotype when \(P\) was expressed. The mutator activity was assessed by screening for auxotrophic mutations arising during Replicative Killing selection for RK\({}^{-}\) clones, or the selection of Rif\({}^{\text{R}}\) mutations within _rpoB_. Since alleles of the host _dnaB_ replicative helicase, or of \(P\), can nullify the mutator phenotype, and since P interacts with DnaB to form an enzymatically inactive \"dead-end\" P-DnaB complex [22], _i.e._, the so-called sequestered DnaB, reducing or eliminating cellular DnaB activity is linked to the mutator phenotype. Under conditions of DnaB sequestration, any replication fork collapse could be problematic for replication re-initiation and generate gaps in DNA synthesis needing repair. Studies reported with yeast suggested that the stalling of transcription at abasic (AP) sites is highly mutagenic [89] and mechanisms, such as mutations arising via translesion synthesis through AP sites were proposed. Most of the auxotrophs we characterized acquired temperature sensitive mutations, characteristic of a missense arising from a point mutation. We did not find that blocking SOS induction, deleting _recA_, or inactivating individual _E. coli_ mutator polymerases Pol IV or Pol V eliminated P-dependent mutagenesis. Since three _E. coli_ DNA polymerases, Pol II, Pol IV and Pol V, are involved in induced mutagenesis [90], this was not evaluated rigorously as the lack of any one could be substituted for by another. Further work is needed to determine if the sequestration of DnaB by P stimulates the appearance of gaps and AP sites around the chromosome, linking them to the mutagenic state associated with elevated levels of P. This would agree with the observation that P can stimulate SOS-independent filamentation [35]. We propose a model that P sequestration of DnaB has several cellular outcomes: (a) it prevents the loading of DnaB helicase needed for the initiation of ColE1 replication (Figure 2) resulting in rapid, complete plasmid curing; (b) it prevents replication restart requiring the reloading of DnaB by DnaC; and (c) when a faster-moving Pol III replisome complex inevitably collides with RNAP, the displaced replisome components along with RNAP from the leading strand are uncoupled from DNA polymerase copying the lagging strand, leading to ssDNA regions that become a target for DNA damage which increase the probability for spontaneous mutation.