DNA replication fidelity in Mycobacterium tuberculosis is mediated by an ancestral prokaryotic proofreader

Abstract

The DNA replication machinery is an important target for antibiotic development in increasingly drug-resistant bacteria, including Mycobacterium tuberculosis. Although blocking DNA replication leads to cell death, disrupting the processes used to ensure replication fidelity can accelerate mutation and the evolution of drug resistance. In Escherichia coli, the proofreading subunit of the replisome, the ɛ exonuclease, is essential for high-fidelity DNA replication; however, we find that the corresponding subunit is completely dispensable in M. tuberculosis. Rather, the mycobacterial replicative polymerase DnaE1 itself encodes an editing function that proofreads DNA replication, mediated by an intrinsic 3'-5' exonuclease activity within its PHP domain. Inactivation of the DnaE1 PHP domain increases the mutation rate by more than 3,000-fold. Moreover, phylogenetic analysis of DNA replication proofreading in the bacterial kingdom suggests that E. coli is a phylogenetic outlier and that PHP domain-mediated proofreading is widely conserved and indeed may be the ancestral prokaryotic proofreader.

Figure 1. The M. tuberculosis DnaE1 polymerase encodes an intrinsic proofreading capability

(A) Rates at which the indicated M. tuberculosis strains acquired resistance to rifampicin were measured by fluctuation analysis. Rv3711c is the annotated dnaQ gene. Circles represent mutant frequency (number of rifampicin-resistant mutants per cell plated in a single culture). Red bars represent the estimated mutation rates (mutations conferring rifampicin resistance per generation), with error bars representing the 95% confidence intervals. (B) Fluctuation analysis was performed with the indicated M. smegmatis strains as in Figure 1A. Ms6275 is the annotated dnaQ gene and Ms4259 is next closest dnaQ homologue. (C) Alignment of DNA polymerase PHP domains from the indicated species. (D) Real-time primer extension activity of purified polymerases. Primer extension results in quenching of template fluorophore. (E) V_max and K_m measurements derived from three primer extension assays. DnaE1^MTB incorporates nucleotides faster than PolIIIα^EC. Data points indicate the mean and error bars the standard deviation. (F) Time course of 3′-5′ exonuclease activity on ssDNA. Wild-type DnaE1^MTB shows robust exonuclease activity while the PHP mutants D23N & D226N do not. Note the distinct digestion patterns of DnaE1^MTB and ε^EC-exonuclease. (G) Primer extension assay as in Figure 2B with a mismatched DNA substrate. Exonuclease deficient polymerases cannot extend from mismatched DNA, while wild-type DnaE1^MTB and PolIIIα^EC+ε^EC activities are unaffected. (H) Gel analysis of primer extension reactions shows that extension from mismatched primers requires exonuclease activity, while extension activity on matched substrates (n.a.) is unaffected.

Figure 2. Inactivation of DnaE1 proofreading results in a mutator phenotype in vivo

(A–B) Fluctuation analysis in M. smegmatis was performed as in Figure 1A. The indicated strains have both the wild-type endogenous dnaE1 allele as well as an anhydrotetracycline (ATc) regulated dnaE1 allele integrated at the L5 attB site. To enable comparison of protein levels, a MYC-tagged dnaE1 allele under the control of its endogenous promoter was loaded under the “WT” lane. For the sake of simplicity, DnaE1^MTB numbering was used throughout the paper. (C) Allele-exchange experiment in a ΔdnaE1 dnaE1::attB(L5) M. smegmatis strain. Plasmids carrying the indicated MYC-tagged dnaE1 alleles were tested for the ability to exchange for the resident attB-integrated plasmid in the parent strain. Error bars indicate standard deviation from three experiments. (D) Growth of indicated M. smegmatis strains.

Figure 3. Conservation of PHP domain-mediated DNA replication proofreading

(A) ε-exonuclease homologues identified by BLAST were compared to the E.coli-like ε-exonuclease dnaQ_proteo (TIGR01406) HMM model. The distribution of scores is shown. (B) Bacterial phylogenetic tree inferred from an alignment of 16S rRNA genes using RAxML. Subsets of eight species from each bacterial class (labeled on the outer ring of the tree) were chosen to represent the total organismal diversity within each class. Species are colored along the outer ring according to the legend as indicated. A subset of PolC containing bacteria (purple strip) have PolC polymerases that have conserved all nine PHP domain metal ion binding residues in addition to having an ε-exonuclease inserted into the PHP domain. For this reason, the PolC containing bacteria have been labeled labeled [‘inactive/active’?].

Figure 4. Inactivation of the PHP domain renders mycobacteria sensitive to nucleoside analogues

(A) Primer extension analysis performed as in Figure 1H in the presence of 200 μM of the adenosine analog ara-A. Incorporation of ara-A impedes primer extension. Whereas wild-type DnaE1^MTB can excise ara-A and resume DNA synthesis, the PHP mutants cannot. (B) Determination of the minimum inhibitory concentration (MIC) of ara-A for the indicated M. smegmatis strains. Pink color indicates cellular respiration; blue color indicates lack of respiration.