Endonuclease Q as a robust enhancer for nucleic acid amplification.

Isothermal nucleic acid amplification techniques are attracting increasing attention in molecular diagnosis and biotechnology. However, most existing techniques are complicated by the need for intricate primer design and numerous enzymes and primers. Here, we have developed a simple method, termed NAQ, that employs adding both endonuclease Q (EndoQ) and dUTP/dITP to conventional rolling circle amplification reactions to increase DNA amplification. NAQ does not require intricate primer design or DNA sequence-specific enzymes, and existing isothermal amplification techniques could be readily adapted to include both EndoQ and dUTP/dITP.

Inhibition of lung microbiota-derived proapoptotic peptides ameliorates acute exacerbation of pulmonary fibrosis.

Idiopathic pulmonary fibrosis is an incurable disease of unknown etiology. Acute exacerbation of idiopathic pulmonary fibrosis is associated with high mortality. Excessive apoptosis of lung epithelial cells occurs in pulmonary fibrosis acute exacerbation. We recently identified corisin, a proapoptotic peptide that triggers acute exacerbation of pulmonary fibrosis. Here, we provide insights into the mechanism underlying the processing and release of corisin. Furthermore, we demonstrate that an anticorisin monoclonal antibody ameliorates lung fibrosis by significantly inhibiting acute exacerbation in the human transforming growth factorß1 model and acute lung injury in the bleomycin model. By investigating the impact of the anticorisin monoclonal antibody in a general model of acute lung injury, we further unravel the potential of corisin to impact such diseases. These results underscore the role of corisin in the pathogenesis of acute exacerbation of pulmonary fibrosis and acute lung injury and provide a novel approach to treating this incurable disease.

Endonuclease V from the archaeon Thermococcus kodakarensis is an inosine-specific ribonuclease.

Endonuclease V (EndoV) is an inosine-specific endonuclease which is highly conserved in all domains of life: Bacteria, Archaea, and Eukarya; and, therefore, may play an important role in nucleic acid processes. It is currently thought that bacterial EndoVs are involved in DNA repair, while eukaryotic EndoVs are involved in RNA editing based on the differences in substrate preferences. However, the role of EndoV proteins, particularly in the archaeal domain, is still poorly understood. Here, we explored the biochemical properties of EndoV from the hyperthermophilic archaeon Thermococcus kodakarensis (TkoEndoV). We show that TkoEndoV has a strong preference for RNA over DNA. Further, we synthesized 1-methylinosine-containing RNA that is a simple TΨC loop mimic of archaeal tRNA and found that TkoEndoV discriminates between 1-methylinosine and inosine, and selectively acts on inosine. Our findings suggest a potential role of archaeal EndoV in the regulation of inosine-containing RNA.

Mutational analysis of Thermococcus kodakarensis Endonuclease III reveals the roles of evolutionarily conserved residues.

Endonuclease III (EndoIII) is nearly ubiquitous in all three domains of life. EndoIII family proteins exhibit a bifunctional (glycosylase/lyase) activity on oxidative/saturated pyrimidine bases, such as thymine glycol. Previous studies on EndoIII homologs have reported the presence of important residues involved in substrate binding and catalytic activity. However, a biochemical clarification of the roles of these residues as well as details of their evolutionary conservation is still lacking. This is particularly true for archaeal orthologs. The current study demonstrated the roles of the evolutionarily conserved residues of euryarchaeon Thermococcus kodakarensis EndoIII (TkoEndoIII). We utilized amino acid sequence analysis and homology modeling to identify highly conserved regions with potential key residues in the EndoIII proteins. Using Ala-substituted TkoEndoIII mutant proteins, residues of interest were quantitatively examined via DNA binding, glycosylase/AP lyase/bifunctional activity, and DNA trapping assays. The obtained results allowed us to determine the roles, as well as the significance of these roles in Schiff base formation (Lys140 as a nucleophile and Asp158), Tg recognition (His160), substrate binding (Arg59, Leu101, Trp102, and Gly136), ß-elimination activities (Ser57 and Asp62), and [4Fe-4S] cluster formation (Cys208 and Cys215). Interestingly, a critical role played by the highly conserved Lys105 (predicted as being away from the catalytic site) in substrate binding, accompanied by a significant indirect effect on catalytic activity, were detected. Our results suggest that these particular residues play conserved roles among EndoIII orthologs across the domains. In addition to identifying the critical role of the highly conserved Lys105, the study provides a comprehensive understanding of the functions attributable to the evolutionarily conserved residues found in the EndoIII family, from Escherichia coli to humans.

Molecular Basis of Substrate Recognition of Endonuclease Q from the Euryarchaeon Pyrococcus furiosus.

Endonuclease Q (EndoQ), a DNA repair endonuclease, was originally identified in the hyperthermophilic euryarchaeon Pyrococcus furiosus in 2015. EndoQ initiates DNA repair by generating a nick on DNA strands containing deaminated bases and an abasic site. Although EndoQ is thought to be important for maintaining genome integrity in certain bacteria and archaea, the underlying mechanism catalyzed by EndoQ remains unclear. Here, we provide insights into the molecular basis of substrate recognition by EndoQ from P. furiosus (PfuEndoQ) using biochemical approaches. Our results of the substrate specificity range and the kinetic properties of PfuEndoQ demonstrate that PfuEndoQ prefers the imide structure in nucleobases along with the discovery of its cleavage activity toward 5,6-dihydrouracil, 5-hydroxyuracil, 5-hydroxycytosine, and uridine in DNA. The combined results for EndoQ substrate binding and cleavage activity analyses indicated that PfuEndoQ flips the target base from the DNA duplex, and the cleavage activity is highly dependent on spontaneous base flipping of the target base. Furthermore, we find that PfuEndoQ has a relatively relaxed substrate specificity; therefore, the role of EndoQ in restriction modification systems was explored. The activity of the EndoQ homolog from Bacillus subtilis was found not to be inhibited by the uracil glycosylase inhibitor from B. subtilis bacteriophage PBS1, whose genome is completely replaced by uracil instead of thymine. Our findings suggest that EndoQ not only has additional functions in DNA repair but also could act as an antiviral enzyme in organisms with EndoQ.IMPORTANCE Endonuclease Q (EndoQ) is a lesion-specific DNA repair enzyme present in certain bacteria and archaea. To date, it remains unclear how EndoQ recognizes damaged bases. Understanding the mechanism of substrate recognition by EndoQ is important to grasp genome maintenance systems in organisms with EndoQ. Here, we find that EndoQ from the euryarchaeon Pyrococcus furiosus recognizes the imide structure in nucleobases by base flipping, and the cleavage activity is enhanced by the base pair instability of the target base, along with the discovery of its cleavage activity toward 5,6-dihydrouracil, 5-hydroxyuracil, 5-hydroxycytosine, and uridine in DNA. Furthermore, a potential role of EndoQ in Bacillus subtilis as an antiviral enzyme by digesting viral genome is demonstrated.

The mesophilic archaeon Methanosarcina acetivorans counteracts uracil in DNA with multiple enzymes: EndoQ, ExoIII, and UDG.

Cytosine deamination into uracil is one of the most prevalent and pro-mutagenic forms of damage to DNA. Base excision repair is a well-known process of uracil removal in DNA, which is achieved by uracil DNA glycosylase (UDG) that is found in all three domains of life. However, other strategies for uracil removal seem to have been evolved in Archaea. Exonuclease III (ExoIII) from the euryarchaeon Methanothermobacter thermautotrophicus has been described to exhibit endonuclease activity toward uracil-containing DNA. Another uracil-acting protein, endonuclease Q (EndoQ), was recently identified from the euryarchaeon Pyrococcus furiosus. Here, we describe the uracil-counteracting system in the mesophilic euryarchaeon Methanosarcina acetivorans through genomic sequence analyses and biochemical characterizations. Three enzymes, UDG, ExoIII, and EndoQ, from M. acetivorans exhibited uracil cleavage activities in DNA with a distinct range of substrate specificities in vitro, and the transcripts for these three enzymes were detected in the M. acetivorans cells. Thus, this organism appears to conduct uracil repair using at least three distinct pathways. Distribution of the homologs of these uracil-targeting proteins in Archaea showed that this tendency is not restricted to M. acetivorans, but is prevalent and diverse in most Archaea. This work further underscores the importance of uracil-removal systems to maintain genome integrity in Archaea, including 'UDG lacking' organisms.

Fluorescence detection of DNA mismatch repair in human cells.

Mismatched base pairs, produced by nucleotide misincorporation by DNA polymerase, are repaired by the mismatch repair (MMR) pathway to maintain genetic integrity. We have developed a method for the fluorescence detection of the cellular MMR ability. A mismatch, which would generate a stop codon in the mRNA transcript unless it was repaired, was introduced into the gene encoding the enhanced green fluorescent protein (EGFP) in an expression plasmid. When MMR-proficient HeLa cells were transformed with this plasmid, the production of active EGFP was observed by fluorescence microscopy. It was assumed that the nick required to initiate the MMR pathway was produced non-specifically in the cells. In contrast, fluorescence was not detected for three types of MMR-deficient cells, LoVo, HCT116, and DLD-1, transformed with the same plasmid. In addition, the expression of a red fluorescent protein gene was utilized to avoid false-negative results. This simple fluorescence method may improve the detection of repair defects, as a biomarker for cancer diagnosis and therapy.

A functional endonuclease Q exists in the bacterial domain: identification and characterization of endonuclease Q from Bacillus pumilus.

DNA base deamination occurs spontaneously under physiological conditions and is promoted by high temperature. Therefore, hyperthermophiles are expected to have efficient repair systems of the deaminated bases in their genomes. Endonuclease Q (EndoQ) was originally identified from the hyperthermophlic archaeon, Pyrococcus furiosus, as a hypoxanthine-specific endonuclease recently. Further biochemical analyses revealed that EndoQ also recognizes uracil, xanthine, and the AP site in DNA, and is probably involved in a specific repair process for damaged bases. Initial phylogenetic analysis showed that an EndoQ homolog is found only in the Thermococcales and some of the methanogens in Archaea, and is not present in most members of the domains Bacteria and Eukarya. A better understanding of the distribution of the EndoQ-mediated repair system is, therefore, of evolutionary interest. We showed here that an EndoQ-like polypeptide from Bacillus pumilus, belonging to the bacterial domain, is functional and has similar properties with the archaeal EndoQs.

PCNA is involved in the EndoQ-mediated DNA repair process in Thermococcales.

To maintain genome integrity for transfer to their offspring, and to maintain order in cellular processes, all living organisms have DNA repair systems. Besides the well-conserved DNA repair machineries, organisms thriving in extreme environments are expected to have developed efficient repair systems. We recently discovered a novel endonuclease, which cleaves the 5' side of deoxyinosine, from the hyperthermophilic archaeon, Pyrococcus furiosus. The novel endonuclease, designated as Endonulcease Q (EndoQ), recognizes uracil, abasic site and xanthine, as well as hypoxanthine, and cuts the phosphodiester bond at their 5' sides. To understand the functional process involving EndoQ, we searched for interacting partners of EndoQ and identified Proliferating Cell Nuclear Angigen (PCNA). The EndoQ activity was clearly enhanced by addition of PCNA in vitro. The physical interaction between the two proteins through a PIP-motif of EndoQ and the toroidal structure of PCNA are critical for the stimulation of the endonuclease activity. These findings provide us a clue to elucidate a unique DNA repair system in Archaea.

EndoQ and EndoV work individually for damaged DNA base repair in Pyrococcus furiosus.

Base deamination is a typical form of DNA damage, and it must be repaired quickly to maintain the genome integrity of living organisms. Endonuclease Q (EndoQ), recently found in the hyperthermophilic archaea, is an enzyme that cleaves the phosphodiester bond 5' from the damaged nucleotide in the DNA strand, and may primarily function to start the repair process for the damaged bases. Endonuclease V (EndoV) also hydrolyzes the second phosphodiester bond 3' from the damaged nucleotide, although the hyperthermophilic archaeal EndoV is a strictly hypoxanthine-specific endonuclease. To understand the relationships of the EndoQ and EndoV functions in hyperthermophilic archaea, we analyzed their interactions in hypoxanthine repair. EndoQ and EndoV do not directly interact with each other in either the presence or absence of DNA. However, EndoQ and EndoV individually worked on deoxyinosine (dI)-containing DNA at each cleavage site. EndoQ has higher affinity to dI-containing DNA than EndoV, and cells produce higher amounts of EndoQ, as compared to EndoV. These data support the proposal that EndoQ primarily functions for, at least, dI-containing DNA.

A novel endonuclease that may be responsible for damaged DNA base repair in Pyrococcus furiosus.

DNA is constantly damaged by endogenous and environmental influences. Deaminated adenine (hypoxanthine) tends to pair with cytosine and leads to the A:T→G:C transition mutation during DNA replication. Endonuclease V (EndoV) hydrolyzes the second phosphodiester bond 3' from deoxyinosine in the DNA strand, and was considered to be responsible for hypoxanthine excision repair. However, the downstream pathway after EndoV cleavage remained unclear. The activity to cleave the phosphodiester bond 5' from deoxyinosine was detected in a Pyrococcus furiosus cell extract. The protein encoded by PF1551, obtained from the mass spectrometry analysis of the purified fraction, exhibited the corresponding cleavage activity. A putative homolog from Thermococcus kodakarensis (TK0887) showed the same activity. Further biochemical analyses revealed that the purified PF1551 and TK0887 proteins recognize uracil, xanthine and the AP site, in addition to hypoxanthine. We named this endonuclease Endonuclease Q (EndoQ), as it may be involved in damaged base repair in the Thermococcals of Archaea.