Selection of dinB alleles suppressing survival loss upon dinB overexpression in Escherichia coli.

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) α-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.

The roles of yeast formins and their regulators Bud6 and Bil2 in the pheromone response.

In response to pheromone Saccharomyces cerevisiae extend a mating projection. This process depends on the formation of polarized actin cables which direct secretion to the mating tip and translocate the nucleus for karyogamy. Here, we demonstrate that proper mating projection formation requires the formin Bni1, as well as the actin nucleation promoting activities of Bud6, but not the formin Bnr1. Further, Bni1 is required for pheromone gradient tracking. Our work also reveals unexpected new functions for Bil2 in the pheromone response. Previously we identified Bil2 as a direct inhibitor of Bnr1 during vegetative cell growth. Here, we show that Bil2 has Bnr1-independent functions in spatially focusing Bni1-GFP at mating projection tips, and in vitro Bil2 and its binding partner Bud6 organize Bni1 into clusters that nucleate actin assembly. bil2∆ cells also display entangled Bni1-generated actin cable arrays and defects in secretory vesicle transport and nuclear positioning. At low pheromone concentrations, bil2∆ cells are delayed in establishing a polarity axis, and at high concentrations they prematurely form a second and a third mating projection. Together, these results suggest that Bil2 promotes the proper formation and timing of mating projections by organizing Bni1 and maintaining a persistent axis of polarized growth.

A single residue unique to DinB-like proteins limits formation of the polymerase IV multiprotein complex in Escherichia coli.

The activity of DinB is governed by the formation of a multiprotein complex (MPC) with RecA and UmuD. We identified two highly conserved surface residues in DinB, cysteine 66 (C66) and proline 67 (P67). Mapping on the DinB tertiary structure suggests these are noncatalytic, and multiple-sequence alignments indicate that they are unique among DinB-like proteins. To investigate the role of the C66-containing surface in MPC formation, we constructed the dinB(C66A) derivative. We found that DinB(C66A) copurifies with its interacting partners, RecA and UmuD, to a greater extent than DinB. Notably, copurification of RecA with DinB is somewhat enhanced in the absence of UmuD and is further increased for DinB(C66A). In vitro pulldown assays also indicate that DinB(C66A) binds RecA and UmuD better than DinB. We note that the increased affinity of DinB(C66A) for UmuD is RecA dependent. Thus, the C66-containing binding surface appears to be critical to modulate interaction with UmuD, and particularly with RecA. Expression of dinB(C66A) from the chromosome resulted in detectable differences in dinB-dependent lesion bypass fidelity and homologous recombination. Study of this DinB derivative has revealed a key surface on DinB, which appears to modulate the strength of MPC binding, and has suggested a binding order of RecA and UmuD to DinB. These findings will ultimately permit the manipulation of these enzymes to deter bacterial antibiotic resistance acquisition and to gain insights into cancer development in humans.

Bil2 Is a Novel Inhibitor of the Yeast Formin Bnr1 Required for Proper Actin Cable Organization and Polarized Secretion.

Cell growth in budding yeast depends on rapid and on-going assembly and turnover of polarized actin cables, which direct intracellular transport of post-Golgi vesicles to the bud tip. Saccharomyces cerevisiae actin cables are polymerized by two formins, Bni1 and Bnr1. Bni1 assembles cables in the bud, while Bnr1 is anchored to the bud neck and assembles cables that specifically extend filling the mother cell. Here, we report a formin regulatory role for YGL015c, a previously uncharacterized open reading frame, which we have named Bud6 Interacting Ligand 2 (BIL2). bil2Δ cells display defects in actin cable architecture and partially-impaired secretory vesicle transport. Bil2 inhibits Bnr1-mediated actin filament nucleation in vitro, yet has no effect on the rate of Bnr1-mediated filament elongation. This activity profile for Bil2 resembles that of another yeast formin regulator, the F-BAR protein Hof1, and we find that bil2Δ with hof1Δ are synthetic lethal. Unlike Hof1, which localizes exclusively to the bud neck, GFP-Bil2 localizes to the cytosol, secretory vesicles, and sites of polarized cell growth. Further, we provide evidence that Hof1 and Bil2 inhibitory effects on Bnr1 are overcome by distinct mechanisms. Together, our results suggest that Bil2 and Hof1 perform distinct yet genetically complementary roles in inhibiting the actin nucleation activity of Bnr1 to control actin cable assembly and polarized secretion.

Integrated control of formin-mediated actin assembly by a stationary inhibitor and a mobile activator.

Formins are essential actin assembly factors whose activities are controlled by a diverse array of binding partners. Until now, most formin ligands have been studied on an individual basis, leaving open the question of how multiple inputs are integrated to regulate formins in vivo. Here, we show that the F-BAR domain of Saccharomyces cerevisiae Hof1 interacts with the FH2 domain of the formin Bnr1 and blocks actin nucleation. Electron microscopy of the Hof1-Bnr1 complex reveals a novel dumbbell-shaped structure, with the tips of the F-BAR holding two FH2 dimers apart. Deletion of Hof1's F-BAR domain in vivo results in disorganized actin cables and secretory defects. The formin-binding protein Bud6 strongly alleviates Hof1 inhibition in vitro, and bud6Δ suppresses hof1Δ defects in vivo. Whereas Hof1 stably resides at the bud neck, we show that Bud6 is delivered to the neck on secretory vesicles. We propose that Hof1 and Bud6 functions are intertwined as a stationary inhibitor and a mobile activator, respectively.

The DinB•RecA complex of Escherichia coli mediates an efficient and high-fidelity response to ubiquitous alkylation lesions.

Alkylation DNA lesions are ubiquitous, and result from normal cellular metabolism as well as from treatment with methylating agents and chemotherapeutics. DNA damage tolerance by translesion synthesis DNA polymerases has an important role in cellular resistance to alkylating agents. However, it is not yet known whether Escherichia coli (E. coli) DNA Pol IV (DinB) alkylation lesion bypass efficiency and fidelity in vitro are similar to those inferred by genetic analyses. We hypothesized that DinB-mediated bypass of 3-deaza-3-methyladenine, a stable analog of 3-methyladenine, the primary replication fork-stalling alkylation lesion, would be of high fidelity. We performed here the first kinetic analyses of E. coli DinB•RecA binary complexes. Whether alone or in a binary complex, DinB inserted the correct deoxyribonucleoside triphosphate (dNTP) opposite either lesion-containing or undamaged template; the incorporation of other dNTPs was largely inefficient. DinB prefers undamaged DNA, but the DinB•RecA binary complex increases its catalytic efficiency on lesion-containing template, perhaps as part of a regulatory mechanism to better respond to alkylation damage. Notably, we find that a DinB derivative with enhanced affinity for RecA, either alone or in a binary complex, is less efficient and has a lower fidelity than DinB or DinB•RecA. This finding contrasts our previous genetic analyses. Therefore, mutagenesis resulting from alkylation lesions is likely limited in cells by the activity of DinB•RecA. These two highly conserved proteins play an important role in maintaining genomic stability when cells are faced with ubiquitous DNA damage. Kinetic analyses are important to gain insights into the mechanism(s) regulating TLS DNA polymerases.