Selection of dinB Alleles Suppressing Survival Loss upon dinB Overexpression in Escherichia coli

Abstract

Escherichia coli strains overproducing DinB undergo survival loss; however, the mechanisms regulating this phenotype are poorly understood. Here we report a genetic selection revealing DinB residues essential to effect this loss-of-survival phenotype. The selection uses strains carrying both an antimutator allele of DNA polymerase III (Pol III) ␣-subunit (dnaE915) and either chromosomal or plasmid-borne dinB alleles. We hypothesized that dnaE915 cells would respond to DinB overproduction differently from dnaE ؉ cells because the dnaE915 allele is known to have an altered genetic interaction with dinB ؉ compared to its interaction with dnaE ؉ . Notably, we observe a loss-of-survival phenotype in dnaE915 strains with either a chromosomal catalytically inactive dinB(D103N) allele or a low-copy-number plasmid-borne dinB ؉ upon DNA damage treatment. Furthermore, we find that the loss-of-survival phenotype occurs independently of DNA damage treatment in a dnaE915 strain expressing the catalytically inactive dinB(D103N) allele from a low-copy-number plasmid. The selective pressure imposed resulted in suppressor mutations that eliminated growth defects. The dinB intragenic mutations examined were either base pair substitutions or those that we inferred to be loss of function (i.e., deletions and insertions). Further analyses of selected novel dinB alleles, generated by single-base-pair substitutions in the dnaE915 strain, indicated that these no longer effect loss of survival upon overproduction in dnaE ؉ strains. These mutations are mapped to specific areas of DinB; this permits us to gain insights into the mechanisms underlying the DinB-mediated overproduction loss-of-survival phenotype.A ll cells accumulate DNA damage that, if left unrepaired, will stall DNA replication due to the inability of high-fidelity DNA polymerases (Pol) to use lesion-containing DNA as templates, a potentially lethal event (1). To ensure survival, Escherichia coli responds to replication fork stalling by upregulating the expression of the SOS-regulated genes (1-3), among which are specialized low-fidelity DNA polymerase genes. These polymerases can perform translesion synthesis (TLS), consisting of both insertion opposite to and elongation from DNA lesions on the template strand (4). TLS can result in elevated mutagenesis. In fact, data suggest that mutations responsible for resistance to several classes of antibiotics require TLS DNA polymerases (5-8). Moreover, these DNA polymerases appear to play a role in the development of disorders such as cancer in metazoans (9-12).E. coli strains lacking the dinB gene (⌬dinB), encoding DinB (DNA Pol IV), one of the TLS DNA polymerases, are sensitive to nitrofurazone (NFZ) and 4-nitroquinoline-1-oxide (4-NQO) (13-15), reagents that generate persistent DNA lesions on the N 2 group of deoxyguanine (N 2 -dG), as well as alkylating agents such as methyl methanesulfonate (MMS) (14,16). Moreover, strains overproducing DinB undergo survival loss. It has been proposed that this is the result of lethal double-s...

DNA polymerase IV primarily operates outside of DNA replication forks in Escherichia coli

“…Punctate foci were visible after ciprofloxacin addition, consistent with pol IV binding to DNA. Cells expressing a catalytically dead variant of pol IV [ 52 , 53 ], DinB(D103N)-YPet, did not produce foci when imaged under the same conditions ( Fig 2C ). We therefore conclude that ciprofloxacin treatment leads to a significant increase in the number of pol IV binding events on the DNA.…”

A Genetic Selection for dinB Mutants Reveals an Interaction between DNA Polymerase IV and the Replicative Polymerase That Is Required for Translesion Synthesis

“…While it remains to be determined whether the reduced rate of replication in response to SOS induction represents a biologically important checkpoint effector function of Pol IV, as previously suggested [ 13 , 45 ], the fact that strains lacking Pol IV function fail to exhibit enhanced sensitivity to agents that generate classes of DNA lesions other than those that Pol IV is capable of bypassing, such as UV photoproducts [ 60 ], argues against such a model. Finally, Benson and colleagues [ 61 ] identified two Pol IV mutants (V7G and F292Y) based on their inability to impede growth of an E . coli strain expressing a mutant Pol IIIα allele ( dnaE915 ).…”

“… Positions of Pol IV mutations identified by Benson et al . [ 61 ] that abrogated lethality caused by overproduced levels of Pol IV in the dnaE915 strain are represented on the in silico model of the Pol IIIαεθ-β clamp-DNA complex in either (A) ribbon or (B) surface views. Residue V7 and F292 of Pol IV are shown in black, while T120 is in orange.…”

RNA Primer Extension Hinders DNA Synthesis by Escherichia coli Mutagenic DNA Polymerase IV

“…It is possible that native DinB undergoes a similar cycle of abortive synthesis with an RNA primer. This mechanism could account for the sickness and lethality that has been observed upon DinB overproduction ( Benson et al, 2014 ).…”