plasmid questions -6

6. Normally multicopy plasmids do not integrate into the chromosome even when they contain considerable DNA sequence homology for recombination. Gutterson and Koshland (1983. Proc. Natl. Acad. Sci. USA 80: 4894-4898) showed that multicopy plasmids (such as pBR322) readily integrate into the chromosome of polA mutants. Suggest an explanation for both of these results.

ANSWER: The polA gene encodes DNA polymerase I, which is required by the cell in tiny amounts for survival but is needed in large quantities by certain plasmid origins to initiate DNA
replication. Thus, a polA(Am) mutation, which is slightly leaky, provides enough DNA PolI for the cell to grow but not enough for plasmids such as pBR322 to replicate autonomously in the cytoplasm. Under this condition the plasmid can be stably inherited only if it integrates into the chromosome via a single cross-over event using regions homologous to the chromosome. In a polA cell the plasmid can autonomously replicate in the cytoplasm, but the homology is still there to allow integration into the chromosome, and this will happen at the same frequency as in the polA mutant. However, the plasmid origin is still functional and expressed at a high level to
increase the copy number of the plasmid. There is a lethal effect because of having a functional plasmid origin disrupting the function of the chromosomal origin and increased copy number could lead to overexpression of chromosomal genes located adjacent to the integrated plasmid, which may also be lethal. So the take home point is that integration still occurs in the polA+ strain, but cells with the integrated plasmid are not recovered because of the lethal consequences.