DNA Metabolism in Balance: Rapid Loss of a RecA-Based Hyperrec Phenotype

Abstract

The RecA recombinase of Escherichia coli has not evolved to optimally promote DNA pairing and strand exchange, the key processes of recombinational DNA repair. Instead, the recombinase function of RecA protein represents an evolutionary compromise between necessary levels of recombinational DNA repair and the potentially deleterious consequences of RecA functionality. A RecA variant, RecA D112R, promotes conjugational recombination at substantially enhanced levels. However, expression of the D112R RecA protein in E. coli results in a reduction in cell growth rates. This report documents the consequences of the substantial selective pressure associated with the RecA-mediated hyperrec phenotype. With continuous growth, the deleterious effects of RecA D112R, along with the observed enhancements in conjugational recombination, are lost over the course of 70 cell generations. The suppression reflects a decline in RecA D112R expression, associated primarily with a deletion in the gene promoter or chromosomal mutations that decrease plasmid copy number. The deleterious effects of RecA D112R on cell growth can also be negated by over-expression of the RecX protein from Neisseria gonorrhoeae. The effects of the RecX proteins in vivo parallel the effects of the same proteins on RecA D112R filaments in vitro. The results indicate that the toxicity of RecA D112R is due to its persistent binding to duplex genomic DNA, creating barriers for other processes in DNA metabolism. A substantial selective pressure is generated to suppress the resulting barrier to growth.

Mechanisms of RecA filament nucleation on ssDNA by the DprA protein

“…In previous model, to assess this impact, the ssDNA binding properties of DprA protein were assayed using EMSA techniques. Previous studies suggested that a homodimer DprA is required for optimal binding to ssDNA, setting the lower limit of oligonucleotide length around 40-50 bases [6, 15, 27].…”

“…Previous findings showed that some hyper-rec events observed for RecA mutants led to a decrease in the yield of recombinants. This suggests that the inhibitory effect of DprA on the yield of recombinants may correspond to its toxic effect on DNA intermediates [15].…”

Design and comparative characterization of RecA variants

“…In fact, RecA did not evolve to have optimized activity but to preserve a balance between different cellular processes and genome stability. Indeed, uncontrolled recombination can interfere with DNA replication and transcription processes 5 . Moreover, bacterial chromosomes frequently carry multiple copies of genes at distinct chromosomal locations, for example, the 7 rrn operons in E. coli, and uncontrolled recombination can cause genetic damage through the aberrant elimination of genomic segments by recombination between repeated sequences 6 .…”

RecA and DNA recombination: a review of molecular mechanisms

“…Actually, recombinogenic over-activity can have deleterious effects on the cell: in fact, noncontrolled recombination can interfere with other biological processes, including DNA replication and transcription. Recombination can also cause genetic damage through the aberrant elimination of genomic segments by the recombination of repeat sequences (9). Hyper-recombinogenic recombinases can be used in vitro for diagnostic applications, particularly in the context of isothermal amplification.…”