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A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded 'mycobacterial mutasome' - minimally comprising DnaE2 polymerase and ImuA' and ImuB accessory proteins - remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III β subunit (β clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuB AAAAGG mutant containing a disrupted β clamp-binding motif abolishes ImuB-β clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this β clamp-binding antibiotic collapses pre-formed ImuB-β clamp complexes. These observations establish the essentiality of the ImuB-β clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance., Competing Interests: SG, ZM, MR, JS, RD, AR, ND, Td, SA, DL, JA, TC, JH, RM, JM, RW, VM, ČV, ML, DW No competing interests declared

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.

Genetic changes lead gradually to altered protein function, making deduction of the molecular basis for activity from a sequence difficult. Comparative studies provide insights into the functional consequences of specific changes. Here we present structural and biochemical studies of NtrC4, a sigma-54 activator from Aquifex aeolicus, and compare it with NtrC1 (a paralog) and NtrC (a homolog from Salmonella enterica) to provide insight into how a substantial change in regulatory mechanism may have occurred. Activity assays show that assembly of NtrC4's active oligomer is repressed by the N-terminal receiver domain, and that BeF3- addition (mimicking phosphorylation) removes this repression. Observation of assembly without activation for NtrC4 indicates that it is much less strongly repressed than NtrC1. The crystal structure of the unactivated receiver-ATPase domain combination shows a partially disrupted interface. NMR structures of the regulatory domain show that its activation mechanism is very similar to that of NtrC1. The crystal structure of the NtrC4 DNA-binding domain shows that it is dimeric and more similar in structure to NtrC than NtrC1. Electron microscope images of the ATPase-DNA-binding domain combination show formation of oligomeric rings. Sequence alignments provide insights into the distribution of activation mechanisms in this family of proteins.

MutS is the key protein of the Escherichia coli DNA mismatch repair system. It recognizes mispaired and unpaired bases and has intrinsic ATPase activity. ATP binding after mismatch recognition by MutS serves as a switch that enables MutL binding and the subsequent initiation of mismatch repair. However, the mechanism of this switch is poorly understood. We have investigated the effects of ATP binding on the MutS structure. Crystallographic studies of ATP-soaked crystals of MutS show a trapped intermediate, with ATP in the nucleotide-binding site. Local rearrangements of several residues around the nucleotide-binding site suggest a movement of the two ATPase domains of the MutS dimer toward each other. Analytical ultracentrifugation experiments confirm such a rearrangement, showing increased affinity between the ATPase domains upon ATP binding and decreased affinity in the presence of ADP. Mutations of specific residues in the nucleotide-binding domain reduce the dimer affinity of the ATPase domains. In addition, ATP-induced release of DNA is strongly reduced in these mutants, suggesting that the two activities are coupled. Hence, it seems plausible that modulation of the affinity between ATPase domains is the driving force for conformational changes in the MutS dimer. These changes are driven by distinct amino acids in the nucleotide-binding site and form the basis for long-range interactions between the ATPase domains and DNA-binding domains and subsequent binding of MutL and initiation of mismatch repair.

We have refined a series of isomorphous crystal structures of the Escherichia coli DNA mismatch repair enzyme MutS in complex with G:T, A:A, C:A and G:G mismatches and also with a single unpaired thymidine. In all these structures, the DNA is kinked by approximately 60 degrees upon protein binding. Two residues widely conserved in the MutS family are involved in mismatch recognition. The phenylalanine, Phe 36, is seen stacking on one of the mismatched bases. The same base is also seen forming a hydrogen bond to the glutamate Glu 38. This hydrogen bond involves the N7 if the base stacking on Phe 36 is a purine and the N3 if it is a pyrimidine (thymine). Thus, MutS uses a common binding mode to recognize a wide range of mismatches.

DNA mismatch repair is an essential safeguard of genomic integrity by removing base mispairings that may arise from DNA polymerase errors or from homologous recombination between DNA strands. In Escherichia coli, the MutS enzyme recognizes mismatches and initiates repair. MutS has an intrinsic ATPase activity crucial for its function, but which is poorly understood. We show here that within the MutS homodimer, the two chemically identical ATPase sites have different affinities for ADP, and the two sites alternate in ATP hydrolysis. A single residue, Arg697, located at the interface of the two ATPase domains, controls the asymmetry. When mutated, the asymmetry is lost and mismatch repair in vivo is impaired. We propose that asymmetry of the ATPase domains is an essential feature of mismatch repair that controls the timing of the different steps in the repair cascade.

DNA mismatch repair ensures genomic integrity on DNA replication. Recognition of a DNA mismatch by a dimeric MutS protein initiates a cascade of reactions and results in repair of the newly synthesized strand; however, details of the molecular mechanism remain controversial. Here we present the crystal structure at 2.2 A of MutS from Escherichia coli bound to a G x T mismatch. The two MutS monomers have different conformations and form a heterodimer at the structural level. Only one monomer recognizes the mismatch specifically and has ADP bound. Mismatch recognition occurs by extensive minor groove interactions causing unusual base pairing and kinking of the DNA. Nonspecific major groove DNA-binding domains from both monomers embrace the DNA in a clamp-like structure. The interleaved nucleotide-binding sites are located far from the DNA. Mutations in human MutS alpha (MSH2/MSH6) that lead to hereditary predisposition for cancer, such as hereditary non-polyposis colorectal cancer, can be mapped to this crystal structure.