Loss of RecA function affects the ability of Escherichia coli to maintain recombinant plasmids containing a Ter site.
In the Escherichia coli chromosome, DNA replication forks arrested by a Tus-Ter complex or by DNA damage are reinitiated through pathways that involve RecA and numerous other recombination functions. To examine the role of recombination in the processing of replication forks arrested by a Tus-Ter complex, the requirements for recombination-associated gene products were assessed in cells carrying Ter plasmids, i.e., plasmids that contain a Ter site oriented to block DNA replication. Of the E. coli recombination functions tested, only loss of recA conferred an observable phenotype on cells containing a Ter plasmid, which was inefficient transformation and reduced ability to maintain a Ter plasmid when Tus was expressed. Given the current understanding of replication reinitiation, the simplest explanation for the restriction of Ter plasmid maintenance was a reduced ability to restart plasmid replication in a recA tus(+) background. However, we were unable to detect a difference in the efficiency of replication arrest by Tus in recA-proficient and recA-deficient cells, which suggests that the inability to restart arrested replication forks is not the cause of the restriction on growth, but is due to an additional function provided by RecA. Other explanations for restriction of Ter plasmid maintenance were examined, including plasmid multimerization, plasmid rearrangements, and copy number differences. The most likely cause of the restriction on Ter plasmid maintenance was a reduced copy number in recA cells that was detected when the copy number was measured in relation to an external control. Possibly, loss of RecA function leads to improper processing of replication forks arrested at a Ter site, leading to the generation of degradation-prone substrates.
Hou
Runhua
R
Department of Microbiology and Immunology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota, 58202-9037
Hill
Thomas M
TM
D004261:DNA Replication; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial / Q000502:physiology*; D010957:Plasmids; D011938:Rec A Recombinases / Q000235:genetics*; D011995:Recombination, Genetic; D020079:Terminal Repeat Sequences; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D011938:Rec A Recombinases