Inefficient replication reduces RecA-mediated repair of UV-damaged plasmids introduced into competent Escherichia coli

Abstract

Transformation of Escherichia coli with purified plasmids containing DNA damage is frequently used as a tool to characterize repair pathways that operate on chromosomes. In this study, we used an assay that allowed us to quantify plasmid survival and to compare how efficiently various repair pathways operate on plasmid DNA introduced into cells relative to their efficiency on chromosomal DNA. We observed distinct differences between the mechanisms operating on the transforming plasmid DNA and the chromosome. An average of one UV-induced lesion was sufficient to inactivate ColE1-based plasmids introduced into nucleotide excision repair mutants, suggesting an essential role for repair on newly introduced plasmid DNA. By contrast, the absence of RecA, RecF, RecBC, RecG or RuvAB had a minimal effect on the survival of the transforming plasmid DNA containing UV-induced damage. Neither the presence of an endogenous homologous plasmid nor the induction of the SOS response enhanced the survival of transforming plasmids. Using two-dimensional agarose-gel analysis, both replication- and RecA-dependent structures that were observed on established, endogenous plasmids following UV-irradiation, failed to form on UV-irradiated plasmids introduced into E. coli. We interpret these observations to suggest that the lack of RecA-mediated survival is likely to be due to inefficient replication that occurs when plasmids are initially introduced into cells, rather than to the plasmid’s size, the absence of homologous sequences, or levels of recA expression.

Events associated with DNA replication disruption are not observed in hydrogen peroxide-treated Escherichia coli

“…Previous studies have shown that replication forks disrupted by UV-induced DNA damage undergo processing events that displace the DNA polymerase and restore the region to its double-stranded form, allowing repair enzymes to access the lesion and effect repair ( Courcelle et al 2003 ; Chow and Courcelle 2004 ; Donaldson et al 2006 ; Belle et al 2007 ; Jeiranian et al 2012 ). The processing forms unique structural intermediates that can be observed using 2D agarose gel electrophoresis.…”

Strand with mutagenic lesion is preferentially used as a template in the region of a bi-stranded clustered DNA damage site in Escherichia coli

“…Another study demonstrated that the inactivation of RecA, RecF or RecG does not affect the survival of transformed plasmids with replication-blocking lesions (i.e. pyrimidine dimers) in a nucleotide-repair-deficient background 34 . The results led the authors to conclude that lesions in the transformed plasmid DNA are repaired within the time gained by an inefficient replication, thus obviating the need for the RecA-dependent tolerance pathway.…”

Inducing a Site Specific Replication Blockage in <i>E. coli</i> Using a Fluorescent Repressor Operator System

“…In vitro analysis has enhanced our understanding of the kinetic behavior of an active replisome when it meets a nucleoprotein blockage 3 , as well as the mechanistic details of the replisome itself 4,5 . Current understanding of the repair of replication is generally undertaken with UV as the damaging agent and studied using plasmid DNA in vivo [6][7][8] . While the proteins that may be involved in repair of DNA after it encounters an in vivo nucleoprotein block are generally understood from these studies, whether there are variations in the molecular events within the repair pathways owing to the distinct cause in the replication block is still yet to be determined.…”