ssb Gene Duplication Restores the Viability of ΔholC and ΔholD Escherichia coli Mutants

Abstract

The HolC-HolD (χψ) complex is part of the DNA polymerase III holoenzyme (Pol III HE) clamp-loader. Several lines of evidence indicate that both leading- and lagging-strand synthesis are affected in the absence of this complex. The Escherichia coli ΔholD mutant grows poorly and suppressor mutations that restore growth appear spontaneously. Here we show that duplication of the ssb gene, encoding the single-stranded DNA binding protein (SSB), restores ΔholD mutant growth at all temperatures on both minimal and rich medium. RecFOR-dependent SOS induction, previously shown to occur in the ΔholD mutant, is unaffected by ssb gene duplication, suggesting that lagging-strand synthesis remains perturbed. The C-terminal SSB disordered tail, which interacts with several E. coli repair, recombination and replication proteins, must be intact in both copies of the gene in order to restore normal growth. This suggests that SSB-mediated ΔholD suppression involves interaction with one or more partner proteins. ssb gene duplication also suppresses ΔholC single mutant and ΔholC ΔholD double mutant growth defects, indicating that it bypasses the need for the entire χψ complex. We propose that doubling the amount of SSB stabilizes HolCD-less Pol III HE DNA binding through interactions between SSB and a replisome component, possibly DnaE. Given that SSB binds DNA in vitro via different binding modes depending on experimental conditions, including SSB protein concentration and SSB interactions with partner proteins, our results support the idea that controlling the balance between SSB binding modes is critical for DNA Pol III HE stability in vivo, with important implications for DNA replication and genome stability.

The inactivation of rfaP, rarA or sspA gene improves the viability of the Escherichia coli DNA polymerase III holD mutant

“…On LB transformants were obtained in the three mutants (Supporting Information Fig. S4B), but, as previously reported (Duigou et al, 2014), holD argE::ssb [pGB-DinB 1 ] clones could not be propagated, in contrast with holD argE::ssb lexAind [pGB-DinB 1 ] clones (Supporting Information Fig. S4C) (which confirms that the lexAind mutation improves the growth of holD argE::ssb [pGB-dinB 1 ] transformants).…”

“…Since HolC contributes to Pol III HE stability on DNA by interacting with SSB at forks, the improvement of the holC mutant viability by a single rfaP or rarA mutation suggests that each of these mutations act, in part, by stabilizing SSB at forks in the absence of HolC. They may promote the conversion of SSB binding mode from SSB 65 to the replicative binding mode SSB 35 , as proposed previously for other holD mutant suppressors, the ssb gene duplication and trk inactivation (Duigou et al ., ; Durand et al ., ). In support with this idea, RarA interacts with SSB (Page et al ., ) and the rfaP mutation perturbs potassium import (see below).…”

“…Two plasmids, pGB2 (vector) and pGB-DinBDC5 that lacks the DinB five last aminoacids and thus does not interact with DnaN were used as controls. (Lenne-Samuel et al, 2002;Duigou et al, 2014). Unfortunately, the holD rfaP lexAind, holD rarA rfaP and holD rarA trkA mutants could not be tested because they were weakly competent and poorly transformed even by the control plasmids.…”

“…Most of the polymerase III holoenzyme subunits are essential for viability, with the notable exception of e, u, v and w (Slater et al, 1994;Saveson and Lovett, 1997;Viguera et al, 2003). In the holD mutant a residual growth allows the appearance of suppressor mutations, restoring holD mutant viability (Viguera et al, 2003;Duigou et al, 2014;Durand et al, 2016). We have assessed the nature of some of these suppressor mutations in our earlier studies.…”

“…Genome sequencing of other suppressed holD mutants showed that a duplication of the ssb gene restores holD mutant growth. We proposed that doubling the amount of SSB protein favors a replicative mode of SSB binding to DNA, which stabilizes HolD-less Pol III HE (Duigou et al, 2014). Finally, mutations that affect potassium import (trkA, trkE) were also found to restore the holD mutant viability, and we proposed that the decrease in intracellular potassium in these mutants strengthens electrostatic interactions between replisome proteins and between replisome and DNA, stabilizing HolD-less Pol III HE enough for efficient replication and viability (Durand et al, 2016).…”