MutS Protein Research Articles

Nucleoside Diphosphate Kinase Escalates A-to-C Mutations in MutT-Deficient Strains of Escherichia coli.

The chemical integrity of the nucleotide pool and its homeostasis are crucial for genome stability. Nucleoside diphosphate kinase (NDK) is a crucial enzyme that carries out reversible conversions from nucleoside diphosphate (NDP) to nucleoside triphosphate (NTP) and deoxynucleoside diphosphate (dNDP) to deoxynucleoside triphosphate (dNTP). Guanosine nucleotides (GDP, GTP, dGDP, and dGTP) are highly susceptible to oxidative damage to 8-oxo-GDP (8-O-GDP), 8-O-dGTP, 8-O-GTP, and 8-O-dGTP. MutT proteins in cells hydrolyze 8-O-GTP to 8-O-GMP or 8-O-dGTP to 8-O-dGMP to avoid its incorporation in nucleic acids. In Escherichia coli, 8-O-dGTP is also known to be hydrolyzed by RibA (GTP cyclohydrolase II). In this study, we show that E. coli NDK catalyzes the conversion of 8-O-dGDP to 8-O-dGTP or vice versa. However, the rate of NDK-mediated phosphorylation of 8-O-dGDP to 8-O-dGTP is about thrice as efficient as the rate of dephosphorylation of 8-O-dGTP to 8-O-dGDP, suggesting an additive role of NDK in net production of 8-O-dGTP in cells. Consistent with this observation, the depletion of NDK (Δndk) in E. coli ΔmutT or ΔmutT ΔribA strains results in a decrease of A-to-C mutations. These observations suggest that NDK contributes to the physiological load of MutT in E. coliIMPORTANCE Nucleoside diphosphate kinase (NDK), a ubiquitous enzyme, is known for its critical role in homeostasis of cellular nucleotide pools. However, NDK has now emerged as a molecule with pleiotropic effects in DNA repair, protein phosphorylation, gene expression, tumor metastasis, development, and pathogen virulence and persistence inside the host. In this study, we reveal an unexpected role of NDK in genome instability because of its activity in converting 8-O-dGDP to 8-O-dGTP. This observation has important consequences in escalating A-to-C mutations in Escherichia coli The severity of NDK in enhancing these mutations may be higher in the organisms challenged with high oxidative stress, which promotes 8-O-dGDP/8-O-dGTP production.

Inactivation of Genes Encoding MutL and MutS Proteins Influences Adhesion and Biofilm Formation by Neisseria gonorrhoeae.

Neisseria gonorrhoeae is an etiological agent of gonorrhea, which remains a global health problem. This bacterium possesses MutL and MutS DNA repair proteins encoded by mutL and mutS genes, whose inactivation causes a mutator phenotype. We have demonstrated the differential gene expression in N. gonorrhoeae mutL and mutS mutants using DNA microarrays. A subset of differentially expressed genes encodes proteins that can influence adhesion and biofilm formation. Compared to the wild-type strain, N. gonorrhoeae mutL and mutS mutants formed denser biofilms with increased biofilm-associated biomass on the abiotic surface. The N. gonorrhoeae mutS::km, but not the mutL mutant, was also more adherent and invasive to human epithelial cells. Further, during infection of epithelial cells with N. gonorrhoeae mutS::km, the expression of some bacterial genes encoding proteins that can influence gonococcal adhesion was changed compared with their expression in cells infected with the wild-type gonococcus, as well as of human genes’ encoding receptors utilized by N. gonorrhoeae (CD46, CEACAM 1, HSPG 2). Thus, deficiency in the mutS gene resulting in increased mutation frequency in singular organisms can be beneficial in populations because these mutants can be a source of features linked to microbial fitness.

Growth and development of AtMSH7 mutants in Arabidopsis thaliana

DNA mismatch repair (MMR) is a highly conserved biological pathway that improves the fidelity of DNA replication and recombination. MMR is initiated when MutS proteins recognize mismatches and small loops of unpaired nucleotides. Arabidopsis thaliana and other plants encode MutS protein homologs (MSH) conserved among other eukaryotic organisms, but also encode an extra MSH polypeptide (MSH7). In order to better understand the role of MSH7 in vivo, a full set of phenotypic parameters that covered the development of the plant from seed imbibition to flowering and seed maturation was analyzed in A. thaliana harboring two different msh7 alleles. Plants deficient in MSH7 show statistically significant faster germination rates, longer primary roots during the juvenile vegetative phase, and higher cauline leaf and axillary and lateral inflorescence numbers compared with wild type. We also quantified number, length and area of siliques and seed number per silique. Disruption of MSH7 resulted in a higher number of smaller siliques than wild type. There were no differences in seed number per silique between genotypes. These findings suggest that mutant plant growth appears to be caused by an impaired cell cycle checkpoint that allows cell division without adequate DNA repair. This increase in proliferation activity demonstrates a functional and temporal link between DNA repair and cell cycle regulation.

Synthesis of β-Diketone DNA Derivatives for Affinity Modification of Proteins

Diketone DNA derivatives have been proposed to modify the guanidine group of Arg in proteins. The β-diketo group at the C2' atom of the sugar phosphate moiety has been introduced in DNA by acylation of oligonucleotide precursors, i.e., DNA fragments containing 2'-amino-2'-deoxyuridine, which have been synthesized by the chemical automatic synthesis. Water-soluble N-[3-(dimethylamino)propyl]-N′-ethylcarbodiimide (EDC) and 4,6-dioxoheptanoic acid have been used in the reaction. The ability of oligodeoxyribonucleotides containing the 2'-β-diketo group to react with guanidine, Nα-Boc-L-arginine, and Nα-Dns-L-arginine has been demonstrated. The introduction of this modification into one of the strands of the 15-base pair DNA duplex has been shown to lead to its destabilization. The conjugate formation of MutS and MutL proteins from the E. coli mismatch repair system with 17-base pair DNA duplexes containing the 2'-deoxy-2'-(4,6-dioxoheptylamido)uridine residue has been detected for the first time. To increase the selectivity of the DNA ligands containing the β-diketo group in the reaction with the Arg residues of proteins, we have proposed to treat the reaction mixture with hydroxylamine. This treatment leads to the cleavage of Schiff bases, which are formed with the involvement of lysine residues.

Single gold-bridged nanoprobes for identification of single point DNA mutations

Consensus ranking of protein affinity to identify point mutations has not been established. Therefore, analytical techniques that can detect subtle variations without interfering with native biomolecular interactions are required. Here we report a rapid method to identify point mutations by a single nanoparticle sensing system. DNA-directed gold crystallization forms rod-like nanoparticles with bridges based on structural design. The nanoparticles enhance Rayleigh light scattering, achieving high refractive-index sensitivity, and enable the system to monitor even a small number of protein-DNA binding events without interference. Analysis of the binding affinity can compile an atlas to distinguish the potential of various point mutations recognized by MutS protein. We use the atlas to analyze the presence and type of single point mutations in BRCA1 from samples of human breast and ovarian cancer cell lines. The strategy of synthesis-by-design of plasmonic nanoparticles for sensors enables direct identification of subtle biomolecular binding distortions and genetic alterations.

Large conformational changes in MutS during DNA scanning, mismatch recognition and repair signaling.

MutS protein recognizes mispaired bases in DNA and targets them for mismatch repair. Little is known about the transient conformations of MutS as it signals initiation of repair. We have used single-molecule fluorescence resonance energy transfer (FRET) measurements to report the conformational dynamics of MutS during this process. We find that the DNA-binding domains of MutS dynamically interconvert among multiple conformations when the protein is free and while it scans homoduplex DNA. Mismatch recognition restricts MutS conformation to a single state. Steady-state measurements in the presence of nucleotides suggest that both ATP and ADP must be bound to MutS during its conversion to a sliding clamp form that signals repair. The transition from mismatch recognition to the sliding clamp occurs via two sequential conformational changes. These intermediate conformations of the MutS:DNA complex persist for seconds, providing ample opportunity for interaction with downstream proteins required for repair.

Toward Single Molecule FRET Studies of DNA Mismatch Repair in Live Bacteria

DNA Mismatch Repair (MMR) is an important correction system that contributes to genomic fidelity during DNA replication. The protein MutS initiates DNA MMR by recognition of base mismatch or insertion/deletion loop and activation of downstream repair processes. Single molecule FRET has previously been used in purified, in vitro studies to characterize conformational transitions in MutS as it interacts with a DNA mismatch and converts to a sliding clamp. The relationship of these conformational transitions to the setting inside live cells remains to be demonstrated. Here we report on efforts to combine in vivo single molecule tracking with single molecule FRET to study the Taq MutS behavior inside live E. coli.

Structural insights into the specificity and catalytic mechanism of mycobacterial nucleotide pool sanitizing enzyme MutT2

Mis-incorporation of modified nucleotides, such as 5-methyl-dCTP or 8-oxo-dGTP, in DNA can be detrimental to genomic integrity. MutT proteins are sanitization enzymes which function by hydrolyzing such nucleotides and regulating the pool of free nucleotides in the cytoplasm. Mycobacterial genomes have a set of four MutT homologs, namely, MutT1, MutT2, MutT3 and MutT4. Mycobacterial MutT2 hydrolyzes 5 m-dCTP and 8-oxo-dGTP to their respective monophosphate products. Additionally, it can hydrolyze canonical nucleotides dCTP and CTP, with a suggested role in sustaining their optimal levels in the nucleotide pool. The structures of M. smegmatis MutT2 and its complexes with cytosine derivatives have been determined at resolutions ranging from 1.10 Å to 1.73 Å. The apo enzyme and its complexes with products (dCMP, CMP and 5 m-dCMP) crystallize in space group P21212, while those involving substrates (dCTP, CTP and 5 m-dCTP) crystallize in space group P21. The molecule takes an α/β/α sandwich fold arrangement, as observed in other MutT homologs. The nucleoside moiety of the ligands is similarly located in all the complexes, while the location of the remaining tail exhibits variability. This is the first report of a MutT2-type protein in complex with ligands. A critical interaction involving Asp116 confers the specificity of the enzyme towards cytosine moieties. A conserved set of enzyme-ligand interactions along with concerted movements of important water molecules provide insights into the mechanism of action.

Suppression of long noncoding RNA NCK1-AS1 increases chemosensitivity to cisplatin in cervical cancer.

Cervical cancer remains a serious health problem till now, with nearly 500,000 women cases diagnosed each year around the world. Long noncoding RNA (lncRNA) is a novel class of RNA transcripts (>200 nucleotides in length) participating in gene transcription, cell proliferation, differentiation, and drug resistance. This study aimed to explore the regulatory relationship among lncRNA NCK1-AS1, miR-134-5p, and MutS protein homolog 2 (MSH2), so that the resistance against cisplatin in cervical cancer treatment could be better understood. Comprehensive lncRNA profiling analysis was performed to screen lncRNAs differentially expressed in cervical cancer. The expression patterns of miR-134-5p, NCK1-AS1, and MSH2 were evaluated in cancerous tissues and adjacent normal tissues obtained from 75 cervical cancer patients. Subsequently, anti-NCK1-AS1 small interfering RNA, miR-134-5p mimics, and miR-134-5p inhibitors were transfected into cervical cancer cells, and the effects of these transcripts on cisplatin resistance and cell apoptosis were investigated. The regulatory relationship among NCK1-AS1, miR-134-5p, and MSH2 was identified using a dual-luciferase reporter gene assay, and the results were further validated by RNA pull-down and RNA immunoprecipitation assays. Based on the microarray data of GSE63514 and GSE27678, NCK1-AS1 was upregulated in cervical cancer. Increased expression of NCK1-AS1, MSH2, and decreased expression of miR-134-5p were observed in cervical cancer tissues. In addition, NCK1-AS1 competitively bound to miR-134-5p to regulate MSH2. Therefore, si-NCK1-AS1 and miR-134-5p mimic both reduced MSH2 activity and increased cisplatin-induced apoptosis in cervical cancer cells. Taken together, NCK1-AS1 may become a novel target in improving the chemotherapeutic response and survival of cervical cancer patients.

Activity of Vsr endonucleases encoded by Neisseria gonorrhoeae FA1090 is influenced by MutL and MutS proteins

BackgroundThe functioning of DNA repair systems is based on correct interactions between proteins involved in DNA repair. Very Short Patch (VSP) repair is a DNA repair system that corrects mismatches resulting from the deamination of 5-methylcytosine. The key enzyme in the VSP system is Vsr endonuclease, which can cleave mismatched DNA independently of accessory proteins. Until now, in vivo activity has only been shown for V.EcoKDcm - the only Vsr endonuclease in Escherichia coli. Additionally, the VSP system of E. coli is the only one for which interactions between proteins of the system have been demonstrated. Neisseria gonorrhoeae FA1090 is the first bacterium that we previously demonstrated to encode two active in vitro Vsr endonucleases: V.NgoAXIII and V.NgoAXIV.ResultsWe elucidate the mutator phenotype of N. gonorrhoeae mutants with disrupted genes encoding V.NgoAXIII or V.NgoAXIV endonuclease. Furthermore, we investigate the interactions between gonococcal Vsr endonucleases and MutL and MutS proteins. The Vsr endonucleases physically interact with gonococcal MutL protein but not with MutS protein. In the presence of the MutL protein, the efficiency of DNA cleavage by both V.NgoAXIII and V.NgoAXIV endonucleases increases, resulting in a decrease in the amount of Vsr enzyme required to complete digestion of mismatched DNA. Both Vsr endonucleases are also stimulated in vitro by the MutL protein of E. coli. In turn, the gonococcal MutS protein hinders DNA cleavage by the Vsr endonucleases. However, this effect is overridden in the presence of MutL, and furthermore, the simultaneous presence of MutL and MutS causes an increase in the efficiency of DNA cleavage by the Vsr endonucleases compared to the reaction catalyzed by V.NgoAXIII or V.NgoAXIV alone.ConclusionsFor the first time, interactions between proteins of the DNA repair system encoded by N. gonorrhoeae that are responsible for the correction of mismatches resulting from the 5-methylcytosine deamination were identified. The increase in activity of Vsr endonucleases in the presence of MutL protein could allow for reduced synthesis of the Vsr endonucleases in cells, and the susceptibility of gonococcal Vsr endonucleases on MutL protein of E. coli implies a universal mechanism of Vsr stimulation by MutL protein.

Specialized Plastids Trigger Tissue-Specific Signaling for Systemic Stress Response in Plants.

Plastids comprise a complex set of organelles in plants that can undergo distinctive patterns of differentiation and redifferentiation during their lifespan. Plastids localized to the epidermis and vascular parenchyma are distinctive in size, structural features, and functions. These plastids are termed "sensory" plastids, and here we show their proteome to be distinct from chloroplasts, with specialized stress-associated features. The distinctive sensory plastid proteome in Arabidopsis (Arabidopsis thaliana) derives from spatiotemporal regulation of nuclear genes encoding plastid-targeted proteins. Perturbation caused by depletion of the sensory plastid-specific protein MutS HOMOLOG1 conditioned local, programmed changes in gene networks controlling chromatin, stress-related phytohormone, and circadian clock behavior and producing a global, systemic stress response in the plant. We posit that the sensory plastid participates in sensing environmental stress, integrating this sensory function with epigenetic and gene expression circuitry to condition heritable stress memory.

Transcriptomic analysis of longitudinal Burkholderia pseudomallei infecting the cystic fibrosis lung

The melioidosis bacterium, Burkholderia pseudomallei, is increasingly being recognised as a pathogen in patients with cystic fibrosis (CF). We have recently catalogued genome-wide variation of paired, isogenic B. pseudomallei isolates from seven Australasian CF cases, which were collected between 4 and 55 months apart. Here, we extend this investigation by documenting the transcriptomic changes in B. pseudomallei in five cases. Following growth in an artificial CF sputum medium, four of the five paired isolates exhibited significant differential gene expression (DE) that affected between 32 and 792 genes. The greatest number of DE events was observed between the strains from patient CF9, consistent with the hypermutator status of the latter strain, which is deficient in the DNA mismatch repair protein MutS. Two patient isolates harboured duplications that concomitantly increased expression of the β-lactamase-encoding gene penA, and a 35 kb deletion in another abolished expression of 29 genes. Convergent expression profiles in the chronically-adapted isolates identified two significantly downregulated and 17 significantly upregulated loci, including the resistance-nodulation-division (RND) efflux pump BpeEF–OprC, the quorum-sensing hhqABCDE operon, and a cyanide- and pyocyanin-insensitive cytochrome bd quinol oxidase. These convergent pathoadaptations lead to increased expression of pathways that may suppress competing bacterial and fungal pathogens, and that enhance survival in oxygen-restricted environments, the latter of which may render conventional antibiotics less effective in vivo. Treating chronically adapted B. pseudomallei infections with antibiotics designed to target anaerobic infections, such as the nitroimidazole class of antibiotics, may significantly improve pathogen eradication attempts by exploiting this Achilles heel.

Endonuclease-independent DNA mismatch repair processes on the lagging strand

DNA mismatch repair (MMR) pathways coordinate the excision and re-synthesis of newly-replicated DNA if a mismatched base-pair has been identified by protein MutS or MutS homologues (MSHs) after replication. DNA excision during MMR is initiated at single-strand breaks (SSBs) in vitro, and several redundant processes have been observed in reconstituted systems which either require a pre-formed SSB in the DNA or require a mismatch-activated nicking endonuclease to introduce a SSB in order to initiate MMR. However, the conditions under which each of these processes may actually occur in living cells have remained obscured by the limitations of current MMR assays. Here we use a novel assay involving chemically-modified oligonucleotide probes to insert targeted DNA ‘mismatches’ directly into the genome of living bacteria to interrogate their replication-coupled repair processes quantitatively in a strand-, orientation-, and mismatched nucleotide-specific manner. This ‘semi-protected oligonucleotide recombination’ (SPORE) assay reveals direct evidence in Escherichia coli of an efficient endonuclease-independent MMR process on the lagging strand—a mechanism that has long-since been considered for lagging-strand repair but never directly shown until now. We find endonuclease-independent MMR is coordinated asymmetrically with respect to the replicating DNA—directed primarily from 3′- of the mismatch—and that repair coordinated from 3′- of the mismatch is in fact the primary mechanism of lagging-strand MMR. While further work is required to explore and identify the molecular requirements for this alternative endonuclease-independent MMR pathway, these findings made possible using the SPORE assay are the first direct report of this long-suspected mechanism in vivo.

Investigating the Link Between Mutations in MutS DNA Repair Protein and Lynch Cancer Syndrome

The Mismatch Repair (MMR) pathway is responsible for correcting errors in DNA and is thus essential for maintaining genome stability in all living organisms. Several studies have shown that mutations causing defects in MMR protein structure and function lead to hypermutation. The increased level of mutations results in serious consequences for human health, highlighted by Lynch Syndrome (LS), a hereditary predisposition to colorectal and other cancers (1). The first protein in the MMR pathway, MutS, recognizes mispaired bases or short insertion‐deletion loops (IDLs) and utilizes its ATPase activity to initiate DNA repair (2). A long‐term study in the Hingorani laboratory examines cancer‐linked single amino acid mutants of the human MutS protein (using Thermus aquaticus MutS as a model system) in order to understand how the changes affect protein structure and function, and alter MMR. This work involves kinetic analysis of MutS activities, and related dynamic conformational changes in the protein as it utilizes ATP to work on DNA. The results will enable construction of a complete mechanism of action for each mutant protein, and help us determine whether the mutants are compromised in their ability to target mismatches, and/or bind and hydrolyze ATP, and/or undergo necessary conformational changes, and therefore understand why they disrupt DNA mismatch repair and give rise to Lynch Syndrome. My project focuses on fluorescence‐based stopped flow kinetic experiments to monitor the conformational changes of wild type MutS and several LS‐linked mutants as they bind +T insertion‐containing DNA and catalyze ATP hydrolysis. In addition to generating new information on each mutant, the findings will advance the proof‐of‐principle concept that detailed analysis of the structure, dynamics, and catalytic activities of individual MutS mutants is both feasible and can reveal critical information for understanding the molecular basis of Lynch Syndrome.Support or Funding InformationNational Institutes of Health R15 GM114743; The Beckman Scholars Program, Arnold and Mabel Beckman FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Accuracy of four mononucleotide-repeat markers for the identification of DNA mismatch-repair deficiency in solid tumors

BackgroundTo screen tumors with microsatellite instability (MSI) arising due to DNA mismatch repair deficiency (dMMR), a panel of five quasi-monomorphic mononucleotide-repeat markers amplified in a multiplex PCR (Pentaplex) are commonly used. In spite of its several strengths, the pentaplex assay is not robust at detecting the loss of MSH6-deficiency (dMSH6). In order to overcome this challenge, we designed this study to develop and optimize a panel of four quasi-monomorphic mononucleotide-repeat markers (Tetraplex) for identifying solid tumors with dMMR, especially dMSH6.MethodsTo improve the sensitivity for tumors with dMMR, we established a quasi-monomorphic variant range (QMVR) of 3–4 bp for the four Tetraplex markers. Thereafter, to confirm the accuracy of this assay, we examined 317 colorectal cancer (CRC) specimens, comprising of 105 dMMR [45 MutL homolog (MLH)1-deficient, 45 MutS protein homolog (MSH)2-deficient, and 15 MSH6-deficient tumors] and 212 MMR-proficient (pMMR) tumors as a test set. In addition, we analyzed a cohort of 138 endometrial cancers (EC) by immunohistochemistry to determine MMR protein expression and validation of our new MSI assay.ResultsUsing the criteria of ≥ 1 unstable markers as MSI-positive tumor, our assay resulted in a sensitivity of 97.1% [95% confidence interval (CI) = 91.9–99.0%] for dMMR, and a specificity of 95.3% (95% CI = 91.5–97.4%) for pMMR CRC specimens. Among the 138 EC specimens, 41 were dMMR according to immunohistochemistry. Herein, our Tetraplex assay detected dMMR tumors with a sensitivity of 92.7% (95% CI = 80.6–97.5%) and a specificity of 97.9% (95% CI = 92.8–99.4%) for pMMR tumors. With respect to tumors with dMSH6, in the CRC-validation set, Tetraplex detected dMSH6 tumors with a sensitivity of 86.7% (13 of 15 dMSH6 CRCs), which was subsequently validated in the EC test set as well (sensitivity, 75.0%; 6 of 8 dMSH6 ECs).ConclusionsOur newly optimized Tetraplex system will help offer a robust and highly sensitive assay for the identification of dMMR in solid tumors.

Loss of CDX2 gene expression is associated with DNA repair proteins and is a crucial member of the Wnt signaling pathway in liver metastasis of colorectal cancer.

Caudal type homeobox 2 (CDX2) has been well-established as a diagnostic marker for colorectal cancer (CRC); however, less is known about its regulation, particularly its potential interactions with the DNA repair proteins, adenomatous polyposis coli (APC) and β-catenin, in a non-transcriptional manner. In the present study, the protein expression of CDX2 was analyzed, depending on the expression of the DNA repair proteins, mismatch repair (MMR), O6-methylguanine DNA methyltransferase (MGMT) and excision repair cross-complementing 1 (ERCC1), and its importance in Wnt signaling was also determined. A total of 101 liver metastases were punched into tissue microarray (TMA) blocks and serial sections were cut for immunohistochemistry. For each protein, an immunoreactive score was generated according to literature data and the scores were fitted to TMA. Subsequently, statistical analysis was performed to compare the levels of expression with each other and with clinical data. CDX2 loss of expression was observed in 38.5% of the CRC liver metastasis cases. A statistically significant association between CDX2 and each of the investigated MMRs was observed: MutL Homolog 1 (P<0.01), MutS protein Homolog (MSH) 2 (P<0.01), MSH6 (P<0.01), and postmeiotic segregation increased 2 (P=0.040). Furthermore, loss of MGMT and ERCC1 was also associated with CDX2 loss (P=0.039 and P<0.01, respectively). In addition, CDX2 and ERCC1 were inversely associated with metastatic tumor size (P=0.038 and P=0.027, respectively). Sustained CDX2 expression was associated with a higher expression of cytoplasmic/membranous β-catenin and with nuclear APC expression (P=0.042 and P<0.01, respectively). In conclusion, CDX2 loss of expression was not a rare event in liver metastasis of CRC and the results suggested that CDX2 may be involved in mechanisms resulting in the loss of DNA repair protein expression, and in turn methylation; however, its exact function in this context remains to be elucidated.

Recombination homeostasis of meiosis during spermatogenesis under nicotine treatment

Cigarette smoking can affect male fertility via the quality of semen. To explore the effects of nicotine, a major component of cigarettes, on meiotic recombination during spermatogenesis, C57BL/6J male mice were injected with nicotine at a dosage of 0.2 mg/100 g body weight daily for 35 days (nicotine-treated group); mice in the control group were injected with isopycnic normal saline. According to previous expression profiles of mouse sperm, a subset of meiosis-related genes was pooled using bioinformatic analysis. Protein expression was compared between the two groups using by Western blotting and immunohistochemistry. Recombination frequency during the meiosis phase of spermatogenesis was estimated by combined use of chromosome spread and immunofluorescence staining in mouse testes. Data mining analysis indicated that 4 genes that express meiotic topoisomerase-like protein SPO11, MutS protein homolog 4 (MSH4), strand exchange protein RAD51 and MutL protein homologue 1 (MLH1), were associated with the meiosis recombination process. The results of Western blotting and immunohistochemistry further showed that the protein expression of SPO11 (0.73-fold) and MSH4 (0.73-fold) was downregulated in murine testes after nicotine treatment, whereas the protein expression of both RAD51 (2.06-fold) and MLH1 (1.40-fold) was upregulated. Unexpectedly, we did not detect a significant difference in recombination frequency in meiosis during spermatogenesis in the nicotine-treated group as compared to the control. Taken together, these results indicate that nicotine can affect the expression profile of restructuring-related genes, but it does not significantly change the recombination frequency during male meiosis. These findings suggest there is a self-regulating mechanism during meiotic chromosome restructuring in male mice that responds to environmental stress. https://doi.org/10.2298/ABS170419025Z Received: April 19, 2017; Revised: May 15, 2017; Accepted: May 18, 2017; Published online: July 26, 2017 How to cite this article: Zhai J, Yuan Q, Yang T, Zhao X, Zhang M, Zhang D, Xu W, Qiao Z. Recombination homeostasis of meiosis during spermatogenesis under nicotine treatment. Arch Biol Sci. 2018;70(1):55-62.

Characterization of the DNA mismatch repair proteins MutS and MutL in a hypermutator Acinetobacter baumannii

Mutations of mutS and mutL genes have been linked with the emergence of hypermutator (HPM) phenotype in several bacteria. Nevertheless, there is scarce evidence that these mutations occurred in HPM Acinetobacter baumannii, therefore, it remains unknown whether the mutations located in domains mediating the functions of MutS and MutL. To address this information gap, the nucleotide sequences of mutS and mutL were characterized and their mutations were identified. Additionally, we proposed in silico models of mutated proteins and analyzed the secondary and tertiary structures, and the interaction interfaces of MutL and MutS. The HPM A. baumannii and a wild-type strain were subjected to PCR amplification of full length mutS and mutL, cloning, and sequencing. Following several reads of both strands of each gene and sequence assembly, the mutations were identified. Thereafter, the three-dimensional (3-D) structure of A. baumannii ATCC 19606 was developed and utilized as a template for homology modeling of the mutated amino acid sequences using the Phyre2 and I-TASSER, VMD 1.9.3, LigPlus v.1.4.5, PyMOL v.0.99 software. Regardless of silent mutations (n = 43), 11 missense mutations were identified in the MutS domains of HPM strain such as A4T, T272S, D278N in N-terminus, connector, and core domains, respectively. Three mutations –I357T, A408S, N447S– and 16 silent mutations were observed in MutL. Secondary structure prediction of MutS revealed that the amount of alpha helices, beta sheets, and coils in HPM were 35, 29, and 63, respectively, while these values were 36, 28, and 63 for A. baumannii ATCC 19606 as non mutator. In the case of MutL, for both HPM and non-mutator, 20, 21, and 39 of complete protein were alpha helices, beta sheets, and coils, respectively. Superimposition of structures of MutS of HPM on non-mutator revealed that T272, D278, G457, S528, A533, Y715, and E747 are closely matched with S272, D278, A457, P528, V533, C715, and K747, respectively in non-mutator strain. When the structure of MutL model in HPM was superimposed on its counterpart in non-mutator, all but residues S447, S408, and T357 were identical. Many mutations along the mutS and mutL were noted, but most of the mutations were observed in the interaction interfaces of MutS and MutL. Other substitutions were predominantly detected in C-terminus of MutS that may lead to reduced ATP binding and hydrolysis. Three substitution mutations were adjacent to C-terminus of MutL and are raising the suggestion of reduction in MutL dimerization. It seems that a combination of these mutations is implicated in increased mutation frequency and accordingly emergence of HPM strain.

Protein expression of Fragile Histidine Triad and cyclooxgenase-2 in serrated neoplasia of the colorectum.

The adenoma-carcinoma sequence (ACS) and the serrated pathway are two distinct developmental routes leading to the formation of colorectal carcinoma (CRC). However, the mechanism triggered by the serrated pathway remains unclear. Therefore, to clarify the molecular and clinicopathological characteristics of the serrated tumorigenic pathway, immunohistochemistry was used to examine the expression of Fragile Histidine Triad (FHIT), cyclooxygenase-2 (COX-2), MutL homolog 1 (MLH1), MutS protein homolog 2 (MSH2) and P53 in endoscopically resected samples of 62 serrated polyps. These samples included 20 hyperplastic polyps (HPs), 16 traditional serrated adenomas (TSAs), 26 sessile serrated adenoma/polyps (SSA/Ps), 20 non-serrated adenomas, 20 carcinoma in adenomas (CIAs) and 18 early pure CRCs without any adenoma component (EPCs). FHIT expression was markedly reduced or absent in 50% of TSA samples, 92.3% of SSA/Ps and 44% of EPCs, but only rarely in HPs, non-serrated adenomas and CIAs. COX-2 expression was more common in non-serrated adenomas compared with in serrated polyps, and was present in 25 and 3.2% of the cases respectively (P<0.01). Furthermore, COX-2 expression was more frequent in CIAs (60%) compared with in EPCs (22.2%; P<0.05). The incidence of negative COX-2 expression was higher in FHIT-negative SSA/Ps compared with in FHIT-positive SSA/Ps (P=0.08). A total of 16.7% of EPC samples and 11.5% of SSA/Ps demonstrated a loss of MLH1/MSH2 expression, but none of the other tumor types did. P53 overexpression was significantly increased in EPC (77.8%) and CIA (60%) samples compared with in HP (0%), TSA (6.6%), SSA/P (0%) and non-serrated adenoma (10%) samples (P<0.01). These findings demonstrated that there are different expression patterns between the serrated pathway and ACS, indicating that aberrant FHIT and inhibited COX-2 expression may be associated with serrated tumorigenesis. In addition, this data indicated that EPC may contain tumors derived from the serrated pathway as well as ACS.

In vitro affinity of Deinococcus radiodurans MutS towards mismatched DNA exceeds that of its orthologues from Escherichia coli and Thermus thermophilus

The mismatch binding protein MutS is responsible for the recognition of mispaired and unpaired bases, which is the initial step in DNA repair. Among the MutS proteins most extensively studied in vitro are those derived from Thermus thermophilus, Thermus aquaticus and Escherichia coli. Here, we present the first report on the in vitro examination of DNA mismatch binding activity of MutS protein from Deinococcus radiodurans and confront this with the properties of those from E. coli and T. thermophilus. The analyses which included mobility gel-shift assay, colorimetric and qPCR estimation of MutS-bound DNA clearly showed that D. radiodurans MutS exhibited much higher affinity towards mismatched DNA in vitro than its counterparts from E. coli and T. thermophilus. In addition, D. radiodurans MutS displayed a significantly higher specificity of DNA mismatch binding than the two other orthologues. The specificity expressed as the ratio of mismatched to fully complementary DNA bound reached over 4 and 20-fold higher values for D. radiodurans than for T. thermophilus and E. coli MutS, respectively. The results demonstrate mainly the biotechnological potential of D. radiodurans MutS but the in vitro characteristics of the MutS orthologues could reflect substantial differences in DNA mismatch binding activities existing in vivo.