Abstract
In bacteria, genetic recombination is a major mechanism for DNA repair. The RecF, RecO and RecR proteins are proposed to initiate recombination by loading the RecA recombinase onto DNA. However, the biophysical mechanisms underlying this process remain poorly understood. Here, we used genetics and single-molecule fluorescence microscopy to investigate whether RecF and RecO function together, or separately, in live Escherichia coli cells. We identified conditions in which RecF and RecO functions are genetically separable. Single-molecule imaging revealed key differences in the spatiotemporal behaviours of RecF and RecO. RecF foci frequently colocalize with replisome markers. In response to DNA damage, colocalization increases and RecF dimerizes. The majority of RecF foci are dependent on RecR. Conversely, RecO foci occur infrequently, rarely colocalize with replisomes or RecF and are largely independent of RecR. In response to DNA damage, RecO foci appeared to spatially redistribute, occupying a region close to the cell membrane. These observations indicate that RecF and RecO have distinct functions in the DNA damage response. The observed localization of RecF to the replisome supports the notion that RecF helps to maintain active DNA replication in cells carrying DNA damage.
Highlights
DNA damage and nucleotide depletion impede DNA replication and occasionally cause single-stranded gaps to be left in the wake of the replisome
Genetic recombination is a major mechanism for DNA repair
The recF, recO and recR genes form a putative epistasis group [5,13,14,15,16,17,18,19,20,21]. This grouping is supported by several findings: (i) an identical level of increased sensitivity to UV irradiation when one of these functions is absent [22]; (ii) almost identical deficiencies in DNA repair and recombination [23]; (iii) the joint suppression of mutant alleles of all three genes by certain mutations in the recA gene [14,24]; and (iv) the existence of a gene in bacteriophage that eliminates the requirement for all three genes in recombination [17,18]
Summary
DNA damage and nucleotide depletion impede DNA replication and occasionally cause single-stranded gaps to be left in the wake of the replisome. The recF, recO and recR genes form a putative epistasis group [5,13,14,15,16,17,18,19,20,21] This grouping is supported by several findings: (i) an identical level of increased sensitivity to UV irradiation when one of these functions is absent [22]; (ii) almost identical deficiencies in DNA repair and recombination [23]; (iii) the joint suppression of mutant alleles of all three genes by certain mutations in the recA gene [14,24]; and (iv) the existence of a gene in bacteriophage that eliminates the requirement for all three genes in recombination [17,18]. Despite extensive examination, evidence for a RecFOR complex––even one formed transiently––is lacking
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