A DinB variant reveals diverse physiological consequences of incomplete TLS extension by a Y-family DNA polymerase

Abstract

The only Y-family DNA polymerase conserved among all domains of life, DinB and its mammalian ortholog pol kappa, catalyzes proficient bypass of damaged DNA in translesion synthesis (TLS). Y-family DNA polymerases, including DinB, have been implicated in diverse biological phenomena ranging from adaptive mutagenesis in bacteria to several human cancers. Complete TLS requires dNTP insertion opposite a replication blocking lesion and subsequent extension with several dNTP additions. Here we report remarkably proficient TLS extension by DinB from Escherichia coli. We also describe a TLS DNA polymerase variant generated by mutation of an evolutionarily conserved tyrosine (Y79). This mutant DinB protein is capable of catalyzing dNTP insertion opposite a replication-blocking lesion, but cannot complete TLS, stalling three nucleotides after an N(2)-dG adduct. Strikingly, expression of this variant transforms a bacteriostatic DNA damaging agent into a bactericidal drug, resulting in profound toxicity even in a dinB(+) background. We find that this phenomenon is not exclusively due to a futile cycle of abortive TLS followed by exonucleolytic reversal. Rather, gene products with roles in cell death and metal homeostasis modulate the toxicity of DinB(Y79L) expression. Together, these results indicate that DinB is specialized to perform remarkably proficient insertion and extension on damaged DNA, and also expose unexpected connections between TLS and cell fate.

Fig. 1.

A pocket of aromatic hydrophobic residues in DinB's active site modulates its TLS function. (A) Structural model of conserved residues in the active site of DinB showing conserved residues (red), the N²-furfuryl moiety (yellow), and the incoming nucleotide (blue). (B) Mutation of Y79 eliminates the ability of DinB to complement NFZ sensitivity. Error bars, the standard deviation determined from three independent cultures.

Fig. 2.

DinB(Y79L) bypasses N²-FG and stalls three nucleotides following the lesion. Primer extension assays with 1, 5, and 50 nM DinB and DinB(Y79L) show that DinB(Y79L) catalyzes lesion bypass, but cannot complete TLS extension. Symbols are as follows: (*) site of N²-FG lesion; (P) radiolabeled primer; (+3) products extended three nucleotides past the lesion; (FL) full-length products. Ratios of stalled to full-length products at 5 and 50 nM enzyme are 120 and 0.2, respectively, for DinB on the undamaged template; 31 and 0.1 for DinB(Y79L) on the undamaged template; 0.9 and 0.02 for DinB on the damaged template; and 107 and 97 for DinB(Y79L) on damaged template.

Fig. 3.

DinB(Y79L) confers hypersensitivity to NFZ and genetic interactions between dinB and dnaQ. (A) Expression of pDinB(Y79L) (closed triangles) or pDinB(F13V) (closed squares) results in profound toxicity to NFZ relative to pDinB (closed circles), or an empty vector control (pWSK29; open circles. Chromosomal genotype is dinB⁺. In this and all graphs in this figure, error bars represent the standard deviation determined from three independent transformants or four independent transductants. (B) Dominance of pDinB(Y79L) and pDinB(F13V) in a ΔdnaQ dinB⁺ background. Symbols and error bars as in A.

Fig. 4.

The toxin-antitoxin module mazEF and the iron import protein tonB modulate the toxicity of DinB(Y79L) and DinB(F13V). (A) Deletion of the toxin-antitoxin (TA) pair mazEF enhances the killing of DinB(Y79L) expression, consistent with a role for mazEF in protection from abortive TLS. Black bars represent the wt strains, and red bars represent ΔmazEF strains. Low NFZ dose is 7.5 μM, high NFZ dose is 12.5 μM. Error bars, standard deviations determined from three independent transformants. Statistical significance was determined by t test (*, P < 0.05; **, P < 0.01; n.s. indicates P > 0.05). (B and C) Deletion of the tonB gene, which encodes an iron import protein, mitigates the toxicity of DinB(Y79L) (in B) and DinB(F13V) expression (in C). Black bars represent wt strains, and red bars represent ΔtonB strains. Symbols otherwise as in A.