Architecture and conservation of the bacterial DNA replication machinery, an underexploited drug target

Abstract

New antibiotics with novel modes of action are required to combat the growing threat posed by multi-drug resistant bacteria. Over the last decade, genome sequencing and other high-throughput techniques have provided tremendous insight into the molecular processes underlying cellular functions in a wide range of bacterial species. We can now use these data to assess the degree of conservation of certain aspects of bacterial physiology, to help choose the best cellular targets for development of new broad-spectrum antibacterials. DNA replication is a conserved and essential process, and the large number of proteins that interact to replicate DNA in bacteria are distinct from those in eukaryotes and archaea; yet none of the antibiotics in current clinical use acts directly on the replication machinery. Bacterial DNA synthesis thus appears to be an underexploited drug target. However, before this system can be targeted for drug design, it is important to understand which parts are conserved and which are not, as this will have implications for the spectrum of activity of any new inhibitors against bacterial species, as well as the potential for development of drug resistance. In this review we assess similarities and differences in replication components and mechanisms across the bacteria, highlight current progress towards the discovery of novel replication inhibitors, and suggest those aspects of the replication machinery that have the greatest potential as drug targets.

Fig. (1)

Architecture and conservation of bacterial replisomes. (A) Bidirectional replication of a circular bacterial chromosome initiates at oriC and terminates opposite. Green circles denote replisomes at replication forks. (B) Model for leading and lagging strand synthesis at a replication fork in E. coli. (C) Overlaid ribbon diagrams of the AAA+ domains of DnaA (PDB: 2HCB, blue), DnaC (PDB: 3ECC, green), DnaI (PDB: 2W58, pink) and Hda (PDB: 3BOS, cyan). The position of the ATP analog AMP-PCP (colored by atom type: C, yellow; N, blue; O, red; P, orange) and a Mg²⁺ ion (gray sphere) within the DnaA structure is shown. (D) Overlaid ribbon diagrams of the AAA+-like domains of the clamp loader subunits τ (blue), δ (magenta), and δ’ (green). Coordinates were derived from PDB: 3GLI. The positions of ADP (colored by atom type, as above for ATP), the phosphate transition state analog BeF₃ (Be, magenta; F, cyan) and a Mg²⁺ ion (gray sphere) within the τ subunit are shown. (E) Phylogenetic tree based on the sequences of DnaC/DnaI helicase loader proteins. The tree was constructed using the neighborhood-joining tree method in Geneious (Biomatters, Auckland, New Zealand), using the Jukes-Cantor genetic distance model and employing the bootstrap method with 100,000 replicates. The sequence of E. coli DnaA was included as an outgroup. Colored boxes indicate helicase loader families (enterobacteria DnaC-type, cyan; Aquificae DnaC-type, pink; firmicute DnaI-type, green). (F) Ribbon diagrams showing filaments of Aquifex aeolicus DnaC (PDB: 3ECC) formed by P6₁ crystal packing [49]. (G) Model for polymerase handover during lagging strand synthesis in Bacillus subtilis [27]. Panels (C), (D) and (F) were created using PyMOL [169].

Fig. (2)

Chemical structures of new inhibitors of DNA gyrase. (A) Simocyclinone D8 [101-105]. (B) GSK299423 [106].

Fig. (3)

DNA polymerase inhibitors. (A) Three-hydrogen-bond interaction between 6-anilinouracil compounds and a cytosine residue within DNA [115]. Chemical structures of (B) new 6-anilinouracil derivatives with improved aqueous solubility [118], (C) a BisQuinol inhibitor of PolC [121], (D) a hybrid PolC/DNA gyrase inhibitor [116], and (E) novel replisome inhibitors identified by high-throughput screening [127].

Fig. (4)

Chemical structures of inhibitors of primosome functions. (A) The DnaB helicase inhibitor, myricetin [129]. (B) A triaminotriazine inhibitor of DnaB helicase [147]. (C) Phenolic saccharide inhibitors of DnaG primase [144]. (D) Sch 642305, an inhibitor of DnaG primase [146]. (E) Inhibitors of DnaG primase produced using structure-aided design [145].

Fig. (5)

Chemical structures of protein-protein interaction inhibitors. (A) RU7, an inhibitor of β-sliding clamp interactions [166]. (B) Inhibitors of interactions mediated by the conserved Cterminal peptide of SSB [168].