Control of Genome Stability by EndoMS/NucS-Mediated Non-Canonical Mismatch Repair

The DNA repair endonuclease EndoMS/NucS is highly conserved in Archaea and Actinobacteria. This enzyme is able to recognize and cleave dsDNA carrying a mismatched base pair, and its activity is enhanced by the interaction with the sliding clamp of the replisome. Today, EndoMS/NucS has been established as the key protein of a non-canonical mismatch repair (MMR) pathway, acting specifically in the repair of transitions and being essential for maintaining genome stability. Despite having some particularities, such as its lower activity on transversions and the inability to correct indels, EndoMS/NucS meets the main hallmarks of a MMR. Its absence leads to a hypermutator phenotype, a transition-biased mutational spectrum and an increase in homeologous recombination. Interestingly, polymorphic EndoMS/NucS variants with a possible effect in mutation rate have been detected in clinical isolates of the relevant actinobacterial pathogen Mycobacterium tuberculosis. Considering that MMR defects are often associated with the emergence of resistant bacteria, the existence of EndoMS/NucS-defective mutators could have an important role in the acquisition of antibiotic resistance in M. tuberculosis. Therefore, a further understanding of the EndoMS/NucS-mediated non-canonical MMR pathway may reveal new strategies to predict and fight drug resistance. This review is focused on the recent progress in NucS, with special emphasis on its effect on genome stability and evolvability in Actinobacteria.

SNP Discrimination by Tolane-Modified Peptide Nucleic Acids: Application for the Detection of Drug Resistance in Pathogens

During the treatment of viral or bacterial infections, it is important to evaluate any resistance to the therapeutic agents used. An amino acid substitution arising from a single base mutation in a particular gene often causes drug resistance in pathogens. Therefore, molecular tools that discriminate a single base mismatch in the target sequence are required for achieving therapeutic success. Here, we synthesized peptide nucleic acids (PNAs) derivatized with tolane via an amide linkage at the N-terminus and succeeded in improving the sequence specificity, even with a mismatched base pair located near the terminal region of the duplex. We assessed the sequence specificities of the tolane-PNAs for single-strand DNA and RNA by UV-melting temperature analysis, thermodynamic analysis, an in silico conformational search, and a gel mobility shift assay. As a result, all of the PNA-tolane derivatives stabilized duplex formation to the matched target sequence without inducing mismatch target binding. Among the different PNA-tolane derivatives, PNA that was modified with a naphthyl-type tolane could efficiently discriminate a mismatched base pair and be utilized for the detection of resistance to neuraminidase inhibitors of the influenza A/H1N1 virus. Therefore, our molecular tool can be used to discriminate single nucleotide polymorphisms that are related to drug resistance in pathogens.

Template and primer requirements for DNA Pol θ-mediated end joining

DNA Pol θ-mediated end joining (TMEJ) is a microhomology-based pathway for repairing double-strand breaks in eukaryotes. TMEJ is also a pathway for nonspecific integration of foreign DNAs into host genomes. DNA Pol θ shares structural homology with the high-fidelity replicases, and its polymerase domain (Polθ) has been shown to extend ssDNA without an apparent template. Using oligonucleotides with distinct sequences, we find that with Mg2+ and physiological salt concentrations, human Polθ has no terminal transferase activity and requires a minimum of 2 bp and optimally 4 bp between a template/primer pair for DNA synthesis. Polθ can tolerate a mismatched base pair at the primer end but loses >90% activity when the mismatch is 2 bp upstream from the active site. Polθ is severely inhibited when the template strand has a 3′ overhang within 3–4 bp from the active site. In line with its TMEJ function, Polθ has limited strand-displacement activity, and the efficiency and extent of primer extension are similar with or without a downstream duplex.

Supercoiling DNA locates mismatches

We present a method of detecting sequence defects by supercoiling DNA with magnetic tweezers. The method is sensitive to a single mismatched base pair in a DNA sequence of several thousand base pairs. We systematically compare DNA molecules with 0 to 16 adjacent mismatches at 1 M monovalent salt and 3.5 pN force and show that, under these conditions, a single plectoneme forms and is stably pinned at the defect. We use these measurements to estimate the energy and degree of end-loop kinking at defects. From this, we calculate the relative probability of plectoneme pinning at the mismatch under physiologically relevant conditions. Based on this estimate, we propose that DNA supercoiling could contribute to mismatch and damage sensing in vivo.

The effects of polymorphisms on human gene targeting

DNA mismatches that occur between vector homology arms and chromosomal target sequences reduce gene targeting frequencies in several species; however, this has not been reported in human cells. Here we demonstrate that even a single mismatched base pair can significantly decrease human gene targeting frequencies. In addition, we show that homology arm polymorphisms can be used to direct allele-specific targeting or to improve unfavorable vector designs that introduce deletions.

The Stability of a Model Substrate for Topoisomerase 1-Mediated DNA Religation Depends on the Presence of Mismatched Base Pairs

Topoisomerase 1 (Top1) enzymes regulate DNA superhelicity by forming covalent cleavage complexes that undergo controlled rotation. Substitution of nucleoside analogs at the +1 position of the DNA duplex relative to the Top1 cleavage site inhibits DNA religation. The reduced efficiency for Top1-mediated religation contributes to the anticancer activity of widely used anticancer drugs including fluoropyrimidines and gemcitabine. In the present study, we report that mismatched base pairs at the +1 position destabilize the duplex DNA components for a model Top1 cleavage complex formation even though one duplex component does not directly include a mismatched base pair. Molecular dynamics simulations reveal G-dU and G-FdU mismatched base pairs, but not a G-T mismatched base pair, increase flexibility at the Top1 cleavage site, and affect coupling between the regions required for the religation reaction to occur. These results demonstrate that substitution of dT analogs into the +1 position of the non-scissile strand alters the stability and flexibility of DNA contributing to the reduced efficiency for Top1-mediated DNA religation. These effects are inherent in the DNA duplex and do not require formation of the Top1:DNA complex. These results provide a biophysical rationale for the inhibition of Top1-mediated DNA religation by nucleotide analog substitution.