NrnA is a 5'-3' exonuclease that processes short RNA substrates in vivo and in vitro.

Bacterial RNases process RNAs until only short oligomers (2-5 nucleotides) remain, which are then processed by one or more specialized enzymes until only nucleoside monophosphates remain. Oligoribonuclease (Orn) is an essential enzyme that acts in this capacity. However, many bacteria do not encode for Orn and instead encode for NanoRNase A (NrnA). Yet, the catalytic mechanism, cellular roles and physiologically relevant substrates have not been fully resolved for NrnA proteins. We herein utilized a common set of reaction assays to directly compare substrate preferences exhibited by NrnA-like proteins from Bacillus subtilis, Enterococcus faecalis, Streptococcus pyogenes and Mycobacterium tuberculosis. While the M. tuberculosis protein specifically cleaved cyclic di-adenosine monophosphate, the B. subtilis, E. faecalis and S. pyogenes NrnA-like proteins uniformly exhibited striking preference for short RNAs between 2-4 nucleotides in length, all of which were processed from their 5' terminus. Correspondingly, deletion of B. subtilis nrnA led to accumulation of RNAs between 2 and 4 nucleotides in length in cellular extracts. Together, these data suggest that many Firmicutes NrnA-like proteins are likely to resemble B. subtilis NrnA to act as a housekeeping enzyme for processing of RNAs between 2 and 4 nucleotides in length.

Overcoming Aggregation-Induced Quenching by Metal-Organic Framework for Electrochemiluminescence (ECL) Enhancement: Zn-PTC as a New ECL Emitter for Ultrasensitive MicroRNAs Detection.

Polycyclic aromatic hydrocarbons (PAHs) as traditional electrochemiluminescence (ECL) luminophores have been widely applied in the analysis field. However, their ECL intensity and efficiency are still limited due to the aggregation-induced quenching (ACQ) effect of PAHs. Hence, to overcome this limitation, we put forward a new strategy to increase the ECL intensity and efficiency by eliminating the ACQ effect of PAHs through the coordinative immobilization of PAHs within metal-organic frameworks (MOFs). As anticipated, the proof-of-concept experiment indicated that the coordinative immobilization of perylene-3,4,9,10-tetracarboxylate (PTC) into a Zn-PTC MOF could distinctly increase the ECL intensity and efficiency compared with H4PTC aggregates and H4PTC monomers. The reason for the ECL enhancement of Zn-PTC was that the immobilization of PTC within the MOF effectively amplified the distance between perylene rings of PTC ligands and thus eliminated the ACQ effect. Furthermore, the PTC into Zn-PTC was stacked in an edge-to-edge mode to form J-aggregation, which was also conducive to ECL enhancement. On the basis of the excellent ECL performance, we utilized Zn-PTC as a new ECL emitter combined with exonuclease III-stimulated target cycling and DNAzyme-assisted cycling dual amplification strategies to construct an ECL sensor for microRNA-21 detection, which had a wide signal response (100 aM to 100 pM) with a detection limit of 29.5 aM. Overall, this work represents a new and convenient method to overcome the ACQ effect of PAHs and boost the ECL performance, which opens a new horizon for developing high-performance ECL materials, thus offering more opportunities for building highly sensitive ECL biosensors.

Turning the Mre11/Rad50 DNA repair complex on its head: lessons from SMC protein hinges, dynamic coiled-coil movements and DNA loop-extrusion?

The bacterial SbcC/SbcD DNA repair proteins were identified over a quarter of a century ago. Following the subsequent identification of the homologous Mre11/Rad50 complex in the eukaryotes and archaea, it has become clear that this conserved chromosomal processing machinery is central to DNA repair pathways and the maintenance of genomic stability in all forms of life. A number of experimental studies have explored this intriguing genome surveillance machinery, yielding significant insights and providing conceptual advances towards our understanding of how this complex operates to mediate DNA repair. However, the inherent complexity and dynamic nature of this chromosome-manipulating machinery continue to obfuscate experimental interrogations, and details regarding the precise mechanisms that underpin the critical repair events remain unanswered. This review will summarize our current understanding of the dramatic structural changes that occur in Mre11/Rad50 complex to mediate chromosomal tethering and accomplish the associated DNA processing events. In addition, undetermined mechanistic aspects of the DNA enzymatic pathways driven by this vital yet enigmatic chromosomal surveillance and repair apparatus will be discussed. In particular, novel and putative models of DNA damage recognition will be considered and comparisons will be made between the modes of action of the Rad50 protein and other related ATPases of the overarching SMC superfamily.

Mutant POLQ and POLZ/REV3L DNA polymerases may contribute to the favorable survival of patients with tumors with POLE mutations outside the exonuclease domain.

BACKGROUND: Mutations in the exonuclease domain of POLE, a DNA polymerase associated with DNA replication and repair, lead to cancers with ultra-high mutation rates. Most studies focus on intestinal and uterine cancers with POLE mutations. These cancers exhibit a significant immune cell infiltrate and favorable prognosis. We questioned whether loss of function of other DNA polymerases can cooperate to POLE to generate the ultramutator phenotype. METHODS: We used cases and data from 15 cancer types in The Cancer Genome Atlas to investigate mutation frequencies of 14 different DNA polymerases. We tested whether tumor mutation burden, patient outcome (disease-free survival) and immune cell infiltration measured by ESTIMATE can be attributed to mutations in POLQ and POLZ/REV3L. RESULTS: Thirty six percent of colorectal, stomach and endometrial cancers with POLE mutations carried additional mutations in POLQ (E/Q), POLZ/REV3L (E/Z) or both DNA polymerases (E/Z/Q). The mutation burden in these tumors was significantly greater compared to POLE-only (E) mutant tumors (p < 0.001). In addition, E/Q, E/Z, and E/Q/Z mutant tumors possessed an increased frequency of mutations in the POLE exonuclease domain (p = 0.013). Colorectal, stomach and endometrial E/Q, E/Z, and E/Q/Z mutant tumors within TCGA demonstrated 100% disease-free survival, even if the POLE mutations occurred outside the exonuclease domain (p = 0.003). However, immune scores in these tumors were related to microsatellite instability (MSI) and not POLE mutation status. This suggests that the host immune response may not be the sole mechanism for prolonged disease-free survival of ultramutated tumors in this cohort. CONCLUSION: Results in this study demonstrate that mutations in POLQ and REV3L in POLE mutant tumors should undergo further investigation to determine whether POLQ and REV3L mutations contribute to the ultramutator phenotype and favorable outcome of patients with POLE mutant tumors.

Label-free and ultrasensitive electrochemical biosensor for the detection of EBV-related DNA based on AgDNCs@DNA/AgNCs nanocomposites and lambda exonuclease-assisted target recycling.

A label-free and efficient electrochemical biosensor was developed for the ultrasensitive detection of EBV-related DNA by combing AgDNCs@DNA/AgNCs nanocomposites with noncanonical lambda exonuclease (λ exo)-assisted target recycling (LNTR). The conjugates of AgDNCs, DNA/AgNCs and probe DNA (pDNA-AgDNCs@DNA/AgNCs conjugates) worked as not only ideal nanocarriers but also efficient electrochemical tags. LNTR didn't require phosphorylated substrates and could be triggered specifically by target DNA, leading to the recycling use of target DNA and the liberation of plentiful linker probes (LP). Subsequently, the LP hybridized with the capture probes on the electrode and then bond to pDNA-AgDNCs@DNA/AgNCs conjugates, generating a sensitive electric signal directly. What's more, the signal amplification effects of DNA/AgNCs and LNTR were investigated. Under the optimal conditions, the proposed method exhibited a wide linear range of 1 fM to 1 nM and the detection limit down to 0.38 fM. In addition, the developed biosensing method exhibited excellent specificity and was successfully applied to detect target DNA in complex biological matrix. The proposed biosensor without extra bio-labels may provide a promising platform in bioanalysis and biochemical research.

Structure and mechanism of the Red recombination system of bacteriophage λ.

While much of this volume focuses on mammalian DNA repair systems that are directly involved in genome stability and cancer, it is important to still be mindful of model systems from prokaryotes. Herein we review the Red recombination system of bacteriophage λ, which consists of an exonuclease for resecting dsDNA ends, and a single-strand annealing protein (SSAP) for binding the resulting 3'-overhang and annealing it to a complementary strand. The genetics and biochemistry of Red have been studied for over 50 years, in work that has laid much of the foundation for understanding DNA recombination in higher eukaryotes. In fact, the Red exonuclease (λ exo) is homologous to Dna2, a nuclease involved in DNA end-resection in eukaryotes, and the Red annealing protein (Redß) is homologous to Rad52, the primary SSAP in eukaryotes. While eukaryotic recombination involves an elaborate network of proteins that is still being unraveled, the phage systems are comparatively simple and streamlined, yet still encompass the fundamental features of recombination, namely DNA end-resection, homologous pairing (annealing), and a coupling between them. Moreover, the Red system has been exploited in powerful methods for bacterial genome engineering that are important for functional genomics and systems biology. However, several mechanistic aspects of Red, particularly the action of the annealing protein, remain poorly understood. This review will focus on the proteins of the Red recombination system, with particular attention to structural and mechanistic aspects, and how the lessons learned can be applied to eukaryotic systems.

Exonuclease domain mutants of yeast DIS3 display genome instability.

The exosome functions to regulate the cellular transcriptome through RNA biogenesis, surveillance, and decay. Mutations in Dis3, a catalytic subunit of the RNA exosome with separable endonuclease and exonuclease activities, are linked to multiple myeloma. Here we report that a cancer-associated DIS3 allele, dis3E729K, provides evidence for DIS3 functioning in mitotic fidelity in yeast. This dis3E729K allele does not induce defects in 7S→5.8S rRNA processing, although it elicits a requirement for P-body function. While it does not significantly influence cell cycle progression alone, the allele reduces the efficiency of cell cycle arrest in strains with defects in kinetochore assembly. Finally, point mutations in the exonuclease domains of yeast Dis3 elicit genome instability phenotypes; however, these DIS3 mutations do not increase DNA damage or RNA processing defects that lead to the accumulation of polyadenylated RNA in the nucleus. These data suggest that specific DIS3 activities support mitotic fidelity in yeast.

The bacterial Mre11-Rad50 homolog SbcCD cleaves opposing strands of DNA by two chemically distinct nuclease reactions.

The Mre11-Rad50 complex is a DNA double-strand break sensor that cleaves blocked DNA ends and hairpins by an ATP-dependent endo/exonuclease activity for subsequent repair. For that, Mre11-Rad50 complexes, including the Escherichia coli homolog SbcCD, can endonucleolytically cleave one or both strands near a protein block and process free DNA ends via a 3'-5' exonuclease, but a unified basis for these distinct activities is lacking. Here we analyzed DNA binding, ATPase and nuclease reactions on different DNA substrates. SbcCD clips terminal bases of both strands of the DNA end in the presence of ATPγS. It introduces a DNA double-strand break around 20-25 bp from a blocked end after multiple rounds of ATP hydrolysis in a reaction that correlates with local DNA meltability. Interestingly, we find that nuclease reactions on opposing strands are chemically distinct, leaving a 5' phosphate on one strand, but a 3' phosphate on the other strand. Collectively, our results identify an unexpected chemical variability of the nuclease, indicating that the complex is oriented at a free DNA end and facing a block with opposite polarity. This suggests a unified model for ATP-dependent endo- and exonuclease reactions at internal DNA near a block and at free DNA ends.

A 3'-5' exonuclease activity embedded in the helicase core domain of Candida albicans Pif1 helicase.

3'-5' exonucleases are frequently found to be associated to polymerases or helicases domains in the same enzyme or could function as autonomous entities. Here we uncovered that Candida albicans Pif1 (CaPif1) displays a 3'-5' exonuclease activity besides its main helicase activity. These two latter activities appear to reside on the same polypeptide and the new exonuclease activity could be mapped to the helicase core domain. We clearly show that CaPif1 displays exclusively exonuclease activity and unambiguously establish the directionality of the exonuclease activity as the 3'-to-5' polarity. The enzyme appears to follow the two-metal-ion driven hydrolyzing activity exhibited by most of the nucleases, as shown by its dependence of magnesium and also by the identification of aspartic residues. Interestingly, an excellent correlation could be found between the presence of the conserved residues and the exonuclease activity when testing activities on Pif1 enzymes from eight fungal organisms. In contrast to others proteins endowed with the double helicase/exonuclease functionality, CaPif1 differs in the fact that the two activities are embedded in the same helicase domain and not located on separated domains. Our findings may suggest a biochemical basis for mechanistic studies of Pif1 family helicases.

Target-protecting dumbbell molecular probe against exonucleases digestion for sensitive detection of ATP and streptavidin.

In this work, a versatile dumbbell molecular (DM) probe was designed and employed in the sensitively homogeneous bioassay. In the presence of target molecule, the DM probe was protected from the digestion of exonucleases. Subsequently, the protected DM probe specifically bound to the intercalation dye and resulted in obvious fluorescence signal which was used to determine the target molecule in return. This design allows specific and versatile detection of diverse targets with easy operation and no sophisticated fluorescence labeling. Integrating the idea of target-protecting DM probe with adenosine triphosphate (ATP) involved ligation reaction, the DM probe with 5'-end phosphorylation was successfully constructed for ATP detection, and the limitation of detection was found to be 4.8 pM. Thanks to its excellent selectivity and sensitivity, this sensing strategy was used to detect ATP spiked in human serum as well as cellular ATP. Moreover, the proposed strategy was also applied in the visual detection of ATP in droplet-based microfluidic platform with satisfactory results. Similarly, combining the principle of target-protecting DM probe with streptavidin (SA)-biotin interaction, the DM probe with 3'-end biotinylation was developed for selective and sensitive SA determination, which demonstrated the robustness and versatility of this design.

Structured RNAs that evade or confound exonucleases: function follows form.

Cells contain powerful RNA decay machinery to eliminate unneeded RNA from the cell, and this process is an important and regulated part of controlling gene expression. However, certain structured RNAs have been found that can robustly resist degradation and extend the lifetime of an RNA. In this review, we present three RNA structures that use a specific three-dimensional fold to provide protection from RNA degradation, and discuss how the recently-solved structures of these RNAs explain their function. Specifically, we describe the Xrn1-resistant RNAs from arthropod-borne flaviviruses, exosome-resistant long non-coding RNAs associated with lung cancer metastasis and found in Kaposi's sarcoma-associated herpesvirus, and tRNA-like sequences occurring in certain plant viruses. These three structures reveal three different mechanisms to protect RNAs from decay and suggest RNA structure-based nuclease resistance may be a widespread mechanism of regulation.

New function of exonuclease and highly sensitive label-free colorimetric DNA detection.

Enzymatic manipulation and modulation of nucleic acids are a central part of cellular function, protection, and reproduction, while rapid and accurate detection of ultralow amount of nucleic acids remains a major challenge in molecular biology research and clinic diagnosis of genetic diseases. Herein, we reported that exonuclease III can degrade the G-quadruplex structure, indicating the new exonuclease's function. Basing on the function of exonuclease III, a novel G-quadruplex-hemin DNAzyme-based colorimetric detection of tumor suppressor gene p53 was successfully developed. Although only one oligonucleotide probe was involved, the sensing strategy could suppress the optical background and achieve an efficient G-quadruplex-hemin DNAzyme-based signal amplification. Specifically, a label-free functional nucleic acid probe (called THzyme probe) was designed via introducing target DNA probe-contained hairpin structure into G-quadruplex DNAzyme. Even if this probe can fold into G-quadruplex structure in the presence of hemin very different from the double-stranded DNA, it is easily degraded by exonuclease III. Thus, no change in UV-vis absorption intensity is detected in the absence of target DNA. However, the hybridization of target DNA can protect the integrity and catalytic activity of THzyme probe, producing the DNAzyme-amplified colorimetric signal. As a result, the p53 gene was able to be detected down to 1.0 pM (final concentration in the signal-generating solution: 50.0 fM) and mismatched target DNAs were easily distinguished. It is expected that this simple sensing methodology for DNA detection can find its utility in point-of-care applications.

Functional Analysis of the Bacteriophage T4 Rad50 Homolog (gp46) Coiled-coil Domain.

Rad50 and Mre11 form a complex involved in the detection and processing of DNA double strand breaks. Rad50 contains an anti-parallel coiled-coil with two absolutely conserved cysteine residues at its apex. These cysteine residues serve as a dimerization domain and bind a Zn(2+) cation in a tetrathiolate coordination complex known as the zinc-hook. Mutation of the zinc-hook in bacteriophage T4 is lethal, indicating the ability to bind Zn(2+) is critical for the functioning of the MR complex. In vitro, we found that complex formation between Rad50 and a peptide corresponding to the C-terminal domain of Mre11 enhances the ATPase activity of Rad50, supporting the hypothesis that the coiled-coil is a major conduit for communication between Mre11 and Rad50. We constructed mutations to perturb this domain in the bacteriophage T4 Rad50 homolog. Deletion of the Rad50 coiled-coil and zinc-hook eliminates Mre11 binding and ATPase activation but does not affect its basal activity. Mutation of the zinc-hook or disruption of the coiled-coil does not affect Mre11 or DNA binding, but their activation of Rad50 ATPase activity is abolished. Although these mutants excise a single nucleotide at a normal rate, they lack processivity and have reduced repetitive exonuclease rates. Restricting the mobility of the coiled-coil eliminates ATPase activation and repetitive exonuclease activity, but the ability to support single nucleotide excision is retained. These results suggest that the coiled-coiled domain adopts at least two conformations throughout the ATPase/nuclease cycle, with one conformation supporting enhanced ATPase activity and processivity and the other supporting nucleotide excision.

A triple-color fluorescent probe for multiple nuclease assays.

We develop a triple-color fluorescent probe which may function as a lab-on-a-DNA-molecule for simultaneous detection of multiple exonucleases/restriction endonucleases. This triple-color fluorescent probe can be further applied for the discrimination of seven exonucleases and four cell lines as well as the screening of various nuclease inhibitors.

Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication.

Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT→TAT and TCG→TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C→A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication.

Identification and structural characterization of two 14-3-3 binding sites in the human peptidylarginine deiminase type VI.

The regulation and function of peptidylarginine deiminase isoform VI (PAD6), which is a highly abundant protein associated with the cytoplasmic lattices in mammalian oocytes, is poorly understood so far. It has been shown previously, that 14-3-3 proteins, a class of regulatory adapter proteins ubiquitous in eukaryotes, bind to PAD6 in vivo in a phosphorylation dependent manner. Here we identify possible 14-3-3 binding sites in human PAD6 by in silico methods, looking for conserved, surface exposed serine residues. Two of these sites were confirmed as 14-3-3 binding sites by fluorescence polarization competition and X-ray crystallography. We furthermore suggest a role of RSK-type kinases in the phosphorylation of one of these two binding sites and provide evidence in the form of in vitro kinase assays with p70S6 kinase and RSK1.

Multiplex targeted proteomic assay for biomarker detection in plasma: a pancreatic cancer biomarker case study.

Biomarkers are most frequently proteins that are measured in the blood. Their development largely relies on antibody creation to test the protein candidate performance in blood samples of diseased versus nondiseased patients. The creation of such antibody assays has been a bottleneck in biomarker progress due to the cost, extensive time, and effort required to complete the task. Targeted proteomics is an emerging technology that is playing an increasingly important role to facilitate disease biomarker development. In this study, we applied a SRM-based targeted proteomics platform to directly detect candidate biomarker proteins in plasma to evaluate their clinical utility for pancreatic cancer detection. The characterization of these protein candidates used a clinically well-characterized cohort that included plasma samples from patients with pancreatic cancer, chronic pancreatitis, and healthy age-matched controls. Three of the five candidate proteins, including gelsolin, lumican, and tissue inhibitor of metalloproteinase 1, demonstrated an AUC value greater than 0.75 in distinguishing pancreatic cancer from the controls. In addition, we provide an analysis of the reproducibility, accuracy, and robustness of the SRM-based proteomics platform. This information addresses important technical issues that could aid in the adoption of the targeted proteomics platform for practical clinical utility.

Crystal structures of aprataxin ortholog Hnt3 reveal the mechanism for reversal of 5'-adenylated DNA.

Aprataxin is a DNA deadenylase that resolves DNA 5'-AMP termini and reverses abortive DNA ligation. The crystal structures of Schizosaccharomyces pombe aprataxin Hnt3 in its apo form and in complex to dsDNA and dsDNA-AMP reveal how Hnt3 recognizes and processes 5'-adenylated DNA in a structure-specific manner. The bound DNA adopts a 5'-flap conformation that facilitates 5'-AMP access to the active site, where AMP cleavage occurs by a canonical catalytic mechanism.

Computational techniques are valuable tools for the discovery of protein-protein interaction inhibitors: the 14-3-3σ case.

Targeting the binding site of 14-3-3 proteins lets the release of partner proteins involved in cell cycle progression, apoptosis, cytoskeletal rearrangement and transcriptional regulation and may therefore be regarded as an alternative strategy to integrate conventional therapeutic approaches against cancer. In the present work, we report the identification of two new small molecule inhibitors of 14-3-3σ/c-Abl protein-protein interaction (BV01 and BV101) discovered by means of computational methods. The most interesting compound (BV01) showed a lethal dose (LD(50)) in the low micromolar range against Ba/F3 murine cell lines expressing the Imatinib (IM)-sensitive wild type Bcr-Abl construct and the IM-resistant Bcr-Abl mutation T315I. BV01 interaction with 14-3-3σ was demonstrated by NMR studies and elucidated by docking. It blocked the binding domain of 14-3-3σ, hence promoting the release of the partner protein c-Abl (the one not involved in Bcr rearrangement), and its translocation to both the nuclear compartment and mitochondrial membranes to induce a pro-apoptotic response. Our results advance BV01 as a confirmed hit compound capable of eliciting apoptotic death of Bcr-Abl-expressing cells by interfering with 14-3-3σ/c-Abl protein-protein interaction.

Critical residue that promotes protein dimerization: a story of partially exposed Phe25 in 14-3-3σ.

Many proteins exist and function as oligomers. While hydrophobic interactions have been recognized as the major driving force for oligomerization, detailed molecular mechanisms for the assembly are unknown. Here, we used 14-3-3σ as a model protein and investigated the role of hydrophobic residues at the dimeric interface using MD simulations and coimmunoprecipitations. We found that a half-exposed and half-buried residue in the interface, Phe(25), plays a more important role in promoting homodimerization than the hydrophobic core residues by organizing both favorable hydrophobic and hydrophilic interactions. Phe(25) is critical in packing and stabilizing hydrophobic core residues. We conclude that the structural stability of hydrophobic cores is critical for a stable homodimer complex and this stable property can be bestowed by residues outside of hydrophobic core. The important organizing activity of Phe(25) for homodimerization of 14-3-3σ originates from its unique physical location, rigidity, size, and hydrophobicity. Thus, hydrophobic residues that are not deeply buried at the oligomeric interface may play important but different roles from the buried core residues and they may promote oligomerization by organizing co-operativity of core and other residues for favorable hydrophobic and electrostatic interactions.