B. subtilis MutS2 splits stalled ribosomes into subunits without mRNA cleavage.

Stalled ribosomes are rescued by pathways that recycle the ribosome and target the nascent polypeptide for degradation. In E. coli, these pathways are triggered by ribosome collisions through the recruitment of SmrB, a nuclease that cleaves the mRNA. In B. subtilis, the related protein MutS2 was recently implicated in ribosome rescue. Here we show that MutS2 is recruited to collisions by its SMR and KOW domains, and we reveal the interaction of these domains with collided ribosomes by cryo-EM. Using a combination of in vivo and in vitro approaches, we show that MutS2 uses its ABC ATPase activity to split ribosomes, targeting the nascent peptide for degradation through the ribosome quality control pathway. However, unlike SmrB, which cleaves mRNA in E. coli, we see no evidence that MutS2 mediates mRNA cleavage or promotes ribosome rescue by tmRNA. These findings clarify the biochemical and cellular roles of MutS2 in ribosome rescue in B. subtilis and raise questions about how these pathways function differently in diverse bacteria.

Regulated Proteolysis of MutSγ Controls Meiotic Crossing Over.

Crossover recombination is essential for accurate chromosome segregation during meiosis. The MutSγ complex, Msh4-Msh5, facilitates crossing over by binding and stabilizing nascent recombination intermediates. We show that these activities are governed by regulated proteolysis. MutSγ is initially inactive for crossing over due to an N-terminal degron on Msh4 that renders it unstable by directly targeting proteasomal degradation. Activation of MutSγ requires the Dbf4-dependent kinase Cdc7 (DDK), which directly phosphorylates and thereby neutralizes the Msh4 degron. Genetic requirements for Msh4 phosphorylation indicate that DDK targets MutSγ only after it has bound to nascent joint molecules (JMs) in the context of synapsing chromosomes. Overexpression studies confirm that the steady-state level of Msh4, not phosphorylation per se, is the critical determinant for crossing over. At the DNA level, Msh4 phosphorylation enables the formation and crossover-biased resolution of double-Holliday Junction intermediates. Our study establishes regulated protein degradation as a fundamental mechanism underlying meiotic crossing over.

FAN1 removes triplet repeat extrusions via a PCNA- and RFC-dependent mechanism.

Human genome-wide association studies have identified FAN1 and several DNA mismatch repair (MMR) genes as modifiers of Huntington's disease age of onset. In animal models, FAN1 prevents somatic expansion of CAG triplet repeats, whereas MMR proteins promote this process. To understand the molecular basis of these opposing effects, we evaluated FAN1 nuclease function on DNA extrahelical extrusions that represent key intermediates in triplet repeat expansion. Here, we describe a strand-directed, extrusion-provoked nuclease function of FAN1 that is activated by RFC, PCNA, and ATP at physiological ionic strength. Activation of FAN1 in this manner results in DNA cleavage in the vicinity of triplet repeat extrahelical extrusions thereby leading to their removal in human cell extracts. The role of PCNA and RFC is to confer strand directionality to the FAN1 nuclease, and this reaction requires a physical interaction between PCNA and FAN1. Using cell extracts, we show that FAN1-dependent CAG extrusion removal relies on a very short patch excision-repair mechanism that competes with MutSß-dependent MMR which is characterized by longer excision tracts. These results provide a mechanistic basis for the role of FAN1 in preventing repeat expansion and could explain the antagonistic effects of MMR and FAN1 in disease onset/progression.

The mismatch recognition protein MutSα promotes nascent strand degradation at stalled replication forks.

Mismatch repair (MMR) is a replication-coupled DNA repair mechanism and plays multiple roles at the replication fork. The well-established MMR functions include correcting misincorporated nucleotides that have escaped the proofreading activity of DNA polymerases, recognizing nonmismatched DNA adducts, and triggering a DNA damage response. In an attempt to determine whether MMR regulates replication progression in cells expressing an ultramutable DNA polymerase ɛ (Polɛ), carrying a proline-to-arginine substitution at amino acid 286 (Polɛ-P286R), we identified an unusual MMR function in response to hydroxyurea (HU)-induced replication stress. Polɛ-P286R cells treated with hydroxyurea exhibit increased MRE11-catalyzed nascent strand degradation. This degradation by MRE11 depends on the mismatch recognition protein MutSα and its binding to stalled replication forks. Increased MutSα binding at replication forks is also associated with decreased loading of replication fork protection factors FANCD2 and BRCA1, suggesting blockage of these fork protection factors from loading to replication forks by MutSα. We find that the MutSα-dependent MRE11-catalyzed fork degradation induces DNA breaks and various chromosome abnormalities. Therefore, unlike the well-known MMR functions of ensuring replication fidelity, the newly identified MMR activity of promoting genome instability may also play a role in cancer avoidance by eliminating rogue cells.

Sorting of mitochondrial and plastid heteroplasmy in Arabidopsis is extremely rapid and depends on MSH1 activity.

The fate of new mitochondrial and plastid mutations depends on their ability to persist and spread among the numerous organellar genome copies within a cell (heteroplasmy). The extent to which heteroplasmies are transmitted across generations or eliminated through genetic bottlenecks is not well understood in plants, in part because their low mutation rates make these variants so infrequent. Disruption of MutS Homolog 1 (MSH1), a gene involved in plant organellar DNA repair, results in numerous de novo point mutations, which we used to quantitatively track the inheritance of single nucleotide variants in mitochondrial and plastid genomes in Arabidopsis. We found that heteroplasmic sorting (the fixation or loss of a variant) was rapid for both organelles, greatly exceeding rates observed in animals. In msh1 mutants, plastid variants sorted faster than those in mitochondria and were typically fixed or lost within a single generation. Effective transmission bottleneck sizes (N) for plastids and mitochondria were N ∼ 1 and 4, respectively. Restoring MSH1 function further increased the rate of heteroplasmic sorting in mitochondria (N ∼ 1.3), potentially because of its hypothesized role in promoting gene conversion as a mechanism of DNA repair, which is expected to homogenize genome copies within a cell. Heteroplasmic sorting also favored GC base pairs. Therefore, recombinational repair and gene conversion in plant organellar genomes can potentially accelerate the elimination of heteroplasmies and bias the outcome of this sorting process.

Antibiotic-induced ROS-mediated Fur allosterism contributes to Helicobacter pylori resistance by inhibiting arsR activation of mutS and mutY.

The increasing antibiotic resistance of Helicobacter pylori primarily driven by genetic mutations poses a significant clinical challenge. Although previous research has suggested that antibiotics could induce genetic mutations in H. pylori, the molecular mechanisms regulating the antibiotic induction remain unclear. In this study, we applied various techniques (e.g., fluorescence microscopy, flow cytometry, and multifunctional microplate reader) to discover that three different types of antibiotics could induce the intracellular generation of reactive oxygen species (ROS) in H. pylori. It is well known that ROS, a critical factor contributing to bacterial drug resistance, not only induces damage to bacterial genomic DNA but also inhibits the expression of genes associated with DNA damage repair, thereby increasing the mutation rate of bacterial genes and leading to drug resistance. However, further research is needed to explore the molecular mechanisms underlying the ROS inhibition of the expression of DNA damage repair-related genes in H. pylori. In this work, we validated that ROS could trigger an allosteric change in the iron uptake regulatory protein Fur, causing its transition from apo-Fur to holo-Fur, repressing the expression of the regulatory protein ArsR, ultimately causing the down-regulation of key DNA damage repair genes (e.g., mutS and mutY); this cascade increased the genomic DNA mutation rate in H. pylori. This study unveils a novel mechanism of antibiotic-induced resistance in H. pylori, providing crucial insights for the prevention and control of antibiotic resistance in H. pylori.

Putative MutS2 Homologs in Algae: More Goods in Shopping Bag?

MutS2 proteins are presumably involved in either control of recombination or translation quality control in bacteria. MutS2 homologs have been found in plants and some algae; however, their actual diversity in eukaryotes remains unknown. We found putative MutS2 homologs in various species of photosynthetic eukaryotes and performed a detailed analysis of the revealed amino acid sequences. Three groups of homologs were distinguished depending on their domain composition: MutS2 homologs with full set of specific domains, MutS2-like sequences without endonuclease Smr domain, and MutS2-like homologs lacking Smr and clamp in domain IV, the extreme form of which are proteins with only a complete ATPase domain. We clarified the information about amino acid composition and set of specific motifs in the conserved domains in MutS2 and MutS2-like sequences. The models of the predicted tertiary structure were obtained for each group of homologs. The phylogenetic analysis demonstrated that all eukaryotic sequences split into two large groups. The first group included homologs belonging to species of Archaeplastida and a subset of haptophyte homologs, while the second-sequences of organisms from CASH groups (cryptophytes, alveolates, stramenopiles, haptophytes) and chlorarachniophytes. The cyanobacterial MutS2 clustered together with the first group, and proteins belonging to Deltaproteobacteria (orders Myxococcales and Bradymonadales) showed phylogenetic affinity to the CASH-including group with strong support. The observed tree pattern did not support a clear differentiation of eukaryotes into lineages with red and green algae-derived plastids. The results are discussed in the context of current conceptions of serial endosymbioses and genetic mosaicism in algae with complex plastids.

A novel homozygote nonsense variant of MSH4 leads to primary ovarian insufficiency and non-obstructive azoospermia.

BACKGROUND: Both non-obstructive azoospermia (NOA) and primary ovarian insufficiency (POI) are pathological conditions characterized by premature and frequently complete gametogenesis failure. Considering that the conserved meiosis I steps are the same between oogenesis and spermatogenesis, inherited defects in meiosis I may result in common causes for both POI and NOA. The present research is a retrospective investigation on an Iranian family with four siblings of both genders who were affected by primary gonadal failure. METHODS: Proband, an individual with NOA, was subjected to clinical examination, hormonal assessment, and genetic consultation. After reviewing the medical history of other infertile members of the family, patients with NOA went through genetic investigations including karyotyping and assessment of Y chromosome microdeletions, followed by Whole exome sequencing (WES) on the proband. After analyzing WES data, the candidate variant was validated using Sanger sequencing and traced in the family. RESULTS: WES analysis of the proband uncovered a novel homozygote nonsense variant, namely c.118C>T in MSH4. This variant resulted in the occurrence of a premature stop codon in residue 40 of MSH4. Notably, the variant was absent in all public exome databases and in the exome data of 400 fertile Iranian individuals. Additionally, the variant was found to co-segregate with infertility in the family. It was also observed that all affected members had homozygous mutations, while their parents were heterozygous and the fertile sister had no mutant allele, corresponding to autosomal recessive inheritance. In addition, we conducted a review of variants reported so far in MSH4, as well as available clinical features related to these variants. The results show that the testicular sperm retrieval and ovarian stimulation cycles have not been successful yet. CONCLUSION: Overall, the results of this study indicate that the identification of pathogenic variants in this gene will be beneficial in selecting proper therapeutic strategies. Also, the findings of this study demonstrate that clinicians should obtain the history of other family members of the opposite sex when diagnosing for POI and/or NOA.

Dynamic human MutSα-MutLα complexes compact mismatched DNA.

DNA mismatch repair (MMR) corrects errors that occur during DNA replication. In humans, mutations in the proteins MutSα and MutLα that initiate MMR cause Lynch syndrome, the most common hereditary cancer. MutSα surveilles the DNA, and upon recognition of a replication error it undergoes adenosine triphosphate-dependent conformational changes and recruits MutLα. Subsequently, proliferating cell nuclear antigen (PCNA) activates MutLα to nick the error-containing strand to allow excision and resynthesis. The structure-function properties of these obligate MutSα-MutLα complexes remain mostly unexplored in higher eukaryotes, and models are predominately based on studies of prokaryotic proteins. Here, we utilize atomic force microscopy (AFM) coupled with other methods to reveal time- and concentration-dependent stoichiometries and conformations of assembling human MutSα-MutLα-DNA complexes. We find that they assemble into multimeric complexes comprising three to eight proteins around a mismatch on DNA. On the timescale of a few minutes, these complexes rearrange, folding and compacting the DNA. These observations contrast with dominant models of MMR initiation that envision diffusive MutS-MutL complexes that move away from the mismatch. Our results suggest MutSα localizes MutLα near the mismatch and promotes DNA configurations that could enhance MMR efficiency by facilitating MutLα nicking the DNA at multiple sites around the mismatch. In addition, such complexes may also protect the mismatch region from nucleosome reassembly until repair occurs, and they could potentially remodel adjacent nucleosomes.

Recurrent mismatch binding by MutS mobile clamps on DNA localizes repair complexes nearby.

DNA mismatch repair (MMR), the guardian of the genome, commences when MutS identifies a mismatch and recruits MutL to nick the error-containing strand, allowing excision and DNA resynthesis. Dominant MMR models posit that after mismatch recognition, ATP converts MutS to a hydrolysis-independent, diffusive mobile clamp that no longer recognizes the mismatch. Little is known about the postrecognition MutS mobile clamp and its interactions with MutL. Two disparate frameworks have been proposed: One in which MutS-MutL complexes remain mobile on the DNA, and one in which MutL stops MutS movement. Here we use single-molecule FRET to follow the postrecognition states of MutS and the impact of MutL on its properties. In contrast to current thinking, we find that after the initial mobile clamp formation event, MutS undergoes frequent cycles of mismatch rebinding and mobile clamp reformation without releasing DNA. Notably, ATP hydrolysis is required to alter the conformation of MutS such that it can recognize the mismatch again instead of bypassing it; thus, ATP hydrolysis licenses the MutS mobile clamp to rebind the mismatch. Moreover, interaction with MutL can both trap MutS at the mismatch en route to mobile clamp formation and stop movement of the mobile clamp on DNA. MutS's frequent rebinding of the mismatch, which increases its residence time in the vicinity of the mismatch, coupled with MutL's ability to trap MutS, should increase the probability that MutS-MutL MMR initiation complexes localize near the mismatch.

New DNA Plasmid Model for Studying DNA Mismatch Repair Response to the G4 Structure.

G-quadruplexes (G4s), the most widely studied alternative DNA structures, are implicated in the regulation of the key cellular processes. In recent years, their involvement in DNA repair machinery has become the subject of intense research. Here, we evaluated the effect of G4 on the prokaryotic DNA mismatch repair (MMR) pathway from two bacterial sources with different mismatch repair mechanisms. The G4 folding, which competes with the maintenance of double-stranded DNA, is known to be controlled by numerous opposing factors. To overcome the kinetic barrier of G4 formation, we stabilized a parallel G4 formed by the d(GGGT)4 sequence in a DNA plasmid lacking a fragment complementary to the G4 motif. Unlike commonly used isolated G4 structures, our plasmid with an embedded stable G4 structure contained elements, such as a MutH cleavage site, required to initiate the repair process. G4 formation in the designed construct was confirmed by Taq polymerase stop assay and dimethyl sulfate probing. The G4-carrying plasmid, together with control ones (lacking a looped area or containing unstructured d(GT)8 insert instead of the G4 motif), were used as new type models to answer the question of whether G4 formation interferes with DNA cleavage as a basic function of MMR.

pilE G-Quadruplex Is Recognized and Preferentially Bound but Not Processed by the MutL Endonuclease from Neisseria gonorrhoeae Mismatch Repair Pathway.

The human pathogen Neisseria gonorrhoeae uses a homologous recombination to undergo antigenic variation and avoid an immune response. The surface protein pilin (PilE) is one of the targets for antigenic variation that can be regulated by N. gonorrhoeae mismatch repair (MMR) and a G-quadruplex (G4) located upstream of the pilE promoter. Using bioinformatics tools, we found a correlation between pilE variability and deletion of DNA regions encoding ngMutS or ngMutL proteins, the main participants in N. gonorrhoeae methyl-independent MMR. To understand whether the G4 structure could affect the ngMutL-mediated regulation of pilin antigenic variation, we designed several synthetic pilE G4-containing oligonucleotides, differing in length, and related DNA duplexes. Using CD measurements and biochemical approaches, we have showed that (i) ngMutL preferentially binds to pilE G4 compared to DNA duplex, although the latter is a cognate substrate for ngMutL endonuclease, (ii) protein binding affinity decreases with shortening of quadruplex-containing and duplex ligands, (iii) the G4 structure inhibits ngMutL-induced DNA nicking and modulates cleavage positions; the enzyme does not cleave DNA within G4, but is able to bypass this noncanonical structure. Thus, pilE G4 may regulate the efficiency of pilin antigenic variation by quadruplex binding to ngMutL and suppression of homologous recombination.

Bioinformatics Analysis of MSH1 Genes of Green Plants: Multiple Parallel Length Expansions, Intron Gains and Losses, Partial Gene Duplications, and Alternative Splicing.

MutS homolog 1 (MSH1) is involved in the recombining and repairing of organelle genomes and is essential for maintaining their stability. Previous studies indicated that the length of the gene varied greatly among species and detected species-specific partial gene duplications in Physcomitrella patens. However, there are critical gaps in the understanding of the gene size expansion, and the extent of the partial gene duplication of MSH1 remains unclear. Here, we screened MSH1 genes in 85 selected species with genome sequences representing the main clades of green plants (Viridiplantae). We identified the MSH1 gene in all lineages of green plants, except for nine incomplete species, for bioinformatics analysis. The gene is a singleton gene in most of the selected species with conserved amino acids and protein domains. Gene length varies greatly among the species, ranging from 3234 bp in Ostreococcus tauri to 805,861 bp in Cycas panzhihuaensis. The expansion of MSH1 repeatedly occurred in multiple clades, especially in Gymnosperms, Orchidaceae, and Chloranthus spicatus. MSH1 has exceptionally long introns in certain species due to the gene length expansion, and the longest intron even reaches 101,025 bp. And the gene length is positively correlated with the proportion of the transposable elements (TEs) in the introns. In addition, gene structure analysis indicated that the MSH1 of green plants had undergone parallel intron gains and losses in all major lineages. However, the intron number of seed plants (gymnosperm and angiosperm) is relatively stable. All the selected gymnosperms contain 22 introns except for Gnetum montanum and Welwitschia mirabilis, while all the selected angiosperm species preserve 21 introns except for the ANA grade. Notably, the coding region of MSH1 in algae presents an exceptionally high GC content (47.7% to 75.5%). Moreover, over one-third of the selected species contain species-specific partial gene duplications of MSH1, except for the conserved mosses-specific partial gene duplication. Additionally, we found conserved alternatively spliced MSH1 transcripts in five species. The study of MSH1 sheds light on the evolution of the long genes of green plants.

Diffuse Large B-Cell Lymphoma, Epstein-Barr Virus -Positive Kappa Monotypic Plasma Cell Proliferation and Invasive Carcinoma, Developing in a Child With Defective Mismatch Repair.

Constitutional mismatch repair deficiency (CMMRD) syndrome is characterized by biallelic mutations in a mismatch repair gene and is associated with development of childhood cancers and symptoms resembling neurofibromatosis type 1, like café-au-lait spots. We describe the extremely rare case of a 12-year-old male presenting with several light brown macular lesions on the skin, gastrointestinal diffuse large B-cell lymphoma, adenomatous polyposis throughout the gastrointestinal tract and an intra-abdominal invasive carcinoma derived from upper gastrointestinal system. All neoplasia, as well as normal tissues, showed loss of Msh6 expression with immunohistochemistry. Molecular studies showed pathogenic homozygous p.F1088Sfs*2 mutation in MSH6. Furthermore, signs consistent with immunodeficiency, namely decreased levels of IgG and IgA in the serum, nodular lymphoid hyperplasia and EBV-associated plasma cell proliferation with monotypic kappa light chain expression in the ileum, were also noted. Our case depicts the phenotypic diversity of CMMRD syndrome and emphasizes its association with immunodeficiency, raising awareness to a feature not widely recognized.

Neisseria gonorrhoeae: DNA Repair Systems and Their Role in Pathogenesis.

Neisseria gonorrhoeae (a Gram-negative diplococcus) is a human pathogen and causative agent of gonorrhea, a sexually transmitted infection. The bacterium uses various approaches for adapting to environmental conditions and multiplying efficiently in the human body, such as regulation of expression of gene expression of surface proteins and lipooligosaccharides (e.g., expression of various forms of pilin). The systems of DNA repair play an important role in the bacterium ability to survive in the host body. This review describes DNA repair systems of N. gonorrhoeae and their role in the pathogenicity of this bacterium. A special attention is paid to the mismatch repair system (MMR) and functioning of the MutS and MutL proteins, as well as to the role of these proteins in regulation of the pilin antigenic variation of the N. gonorrhoeae pathogen.

Direct Detection of Low Abundance Genes of Single Point Mutation.

Cell-free DNA (cfDNA) analysis, specifically circulating tumor DNA (ctDNA) analysis, provides enormous opportunities for noninvasive early assessment of cancers. To date, PCR-based methods have led this field. However, the limited sensitivity/specificity of PCR-based methods necessitates the search for new methods. Here, we describe a direct approach to detect KRAS G12D mutated genes in clinical ctDNA samples with the utmost LOD and sensitivity/specificity. In this study, MutS protein was immobilized on the tip of an atomic force microscope (AFM), and the protein sensed the mismatched sites of the duplex formed between the capture probe on the surface and mutated DNA. A noteworthy LOD (3 copies, 0.006% allele frequency) was achieved, along with superb sensitivity/specificity (100%/100%). These observations demonstrate that force-based AFM, in combination with the protein found in nature and properly designed capture probes/blockers, represents an exciting new avenue for ctDNA analysis.

Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA-Protein Complexes Using Mismatch Repair Protein MutS as a Model.

Covalent protein capture (cross-linking) by reactive DNA derivatives makes it possible to investigate structural features by fixing complexes at different stages of DNA-protein recognition. The most common cross-linking methods are based on reactive groups that interact with native or engineered cysteine residues. Nonetheless, high reactivity of most of such groups leads to preferential fixation of early-stage complexes or even non-selective cross-linking. We synthesised a set of DNA reagents carrying an acrylamide group attached to the C5 atom of a 2'-deoxyuridine moiety via various linkers and studied cross-linking with MutS as a model protein. MutS scans DNA for mismatches and damaged nucleobases and can form multiple non-specific complexes with DNA that may cause non-selective cross-linking. By varying the length of the linker between DNA and the acrylamide group and by changing the distance between the reactive nucleotide and a mismatch in the duplex, we showed that cross-linking occurs only if the distance between the acrylamide group and cysteine is optimal within the DNA-protein complex. Thus, acrylamide-modified DNA duplexes are excellent tools for studying DNA-protein interactions because of high selectivity of cysteine trapping.

Complex Evolution of the Mismatch Repair System in Eukaryotes is Illuminated by Novel Archaeal Genomes.

Repairing DNA damage is one of the most important functions of the 'housekeeping' proteins, as DNA molecules are constantly subject to different kinds of damage. An important mechanism of DNA repair is the mismatch repair system (MMR). In eukaryotes, it is more complex than it is in bacteria or Archaea due to an inflated number of paralogues produced as a result of an extensive process of gene duplication and further specialization upon the evolution of the first eukaryotes, including an important part of the meiotic machinery. Recently, the discovery and sequencing of Asgard Archaea allowed us to revisit the MMR system evolution with the addition of new data from a group that is closely related to the eukaryotic ancestor. This new analysis provided evidence for a complex evolutionary history of eukaryotic MMR: an archaeal origin for the nuclear MMR system in eukaryotes, with subsequent acquisitions of other MMR systems from organelles.

The methylation-independent mismatch repair machinery in Pseudomonas aeruginosa.

Over the last 70 years, we've all gotten used to an Escherichia coli-centric view of the microbial world. However, genomics, as well as the development of improved tools for genetic manipulation in other species, is showing us that other bugs do things differently, and that we cannot simply extrapolate from E. coli to everything else. A particularly good example of this is encountered when considering the mechanism(s) involved in DNA mismatch repair by the opportunistic human pathogen, Pseudomonas aeruginosa (PA). This is a particularly relevant phenotype to examine in PA, since defects in the mismatch repair (MMR) machinery often give rise to the property of hypermutability. This, in turn, is linked with the vertical acquisition of important pathoadaptive traits in the organism, such as antimicrobial resistance. But it turns out that PA lacks some key genes associated with MMR in E. coli, and a closer inspection of what is known (or can be inferred) about the MMR enzymology reveals profound differences compared with other, well-characterized organisms. Here, we review these differences and comment on their biological implications.

MutS protein-based fiber optic particle plasmon resonance biosensor for detecting single nucleotide polymorphisms.

A new biosensing method is presented to detect gene mutation by integrating the MutS protein from bacteria with a fiber optic particle plasmon resonance (FOPPR) sensing system. In this method, the MutS protein is conjugated with gold nanoparticles (AuNPs) deposited on an optical fiber core surface. The target double-stranded DNA containing an A and C mismatched base pair in a sample can be captured by the MutS protein, causing increased absorption of green light launching into the fiber and hence a decrease in transmitted light intensity through the fiber. As the signal change is enhanced through consecutive total internal reflections along the fiber, the limit of detection for an AC mismatch heteroduplex DNA can be as low as 0.49 nM. Because a microfluidic chip is used to contain the optical fiber, the narrow channel width allows an analysis time as short as 15 min. Furthermore, the label-free and real-time nature of the FOPPR sensing system enables determination of binding affinity and kinetics between MutS and single-base mismatched DNA. The method has been validated using a heterozygous PCR sample from a patient to determine the allelic fraction. The obtained allelic fraction of 0.474 reasonably agrees with the expected allelic fraction of 0.5. Therefore, the MutS-functionalized FOPPR sensor may potentially provide a convenient quantitative tool to detect single nucleotide polymorphisms in biological samples with a short analysis time at the point-of-care sites.