Arabidopsis thaliana endonuclease V is a ribonuclease specific for inosine-containing single-stranded RNA

Endonuclease V is highly conserved, both structurally and functionally, from bacteria to humans, and it cleaves the deoxyinosine-containing double-stranded DNA in Escherichia coli , whereas in Homo sapiens it catalyses the inosine-containing single-stranded RNA. Thus, deoxyinosine and inosine are unexpectedly produced by the deamination reactions of adenine in DNA and RNA, respectively. Moreover, adenosine-to-inosine (A-to-I) RNA editing is carried out by adenosine deaminase acting on dsRNA (ADARs). We focused on Arabidopsis thaliana endonuclease V (AtEndoV) activity exhibiting variations in DNA or RNA substrate specificities. Since no ADAR was observed for A-to-I editing in A. thaliana , the possibility of inosine generation by A-to-I editing can be ruled out. Purified AtEndoV protein cleaved the second and third phosphodiester bonds, 3′ to inosine in single-strand RNA, at a low reaction temperature of 20–25°C, whereas the AtEndoV (Y100A) protein bearing a mutation in substrate recognition sites did not cleave these bonds. Furthermore, AtEndoV, similar to human EndoV, prefers RNA substrates over DNA substrates, and it could not cleave the inosine-containing double-stranded RNA. Thus, we propose the possibility that AtEndoV functions as an RNA substrate containing inosine induced by RNA damage, and not by A-to-I RNA editing in vivo .

A Hyperthermophilic Argonaute From Ferroglobus placidus With Specificity on Guide Binding Pattern

Argonaute proteins (Agos) from thermophilic archaea are involved in several important processes, such as host defense and DNA replication. The catalytic mechanism of Ago from different microbes with great diversity and genome editing potential is attracting increasing attention. Here, we describe an Argonaute from hyperthermophilic Ferroglobus placidus (FpAgo), with a typical DNA-guided DNA endonuclease activity but adopted with only a short guide 15–20 nt length rather than a broad guide selectivity for reported Agos. FpAgo performed the precise cleavage of phosphodiester bonds between 10 and 11 nt on the target strand (counting from the guide strand) guided strictly by 5′-phosphorylated DNA at temperatures ranging from 75 to 99°C. The cleavage activity was regulated by the divalent cations Mn2+, Mg2+, Co2+, and Ni2+. In addition, FpAgo possesses guide/target mismatch tolerance in the seed region but is sensitive to mismatches in the 3′-guide region. Notably, the EMSA assay revealed that the FpAgo-guide-target ternary complex exhibited a stronger binding affinity for short 15 and 16 nt guide DNAs than longer guides. Moreover, we performed structural modeling analyses that implied the unique PAZ domain of FpAgo for 3′-guide recognition and binding to affect guide length specificity. This study broadens our understanding of thermophilic Agos and paves the way for their use in DNA manipulation.

Functional and LC-MS/MS analysis of in vitro transcribed mRNAs carrying phosphorothioate or boranophosphate moieties reveal polyA tail modifications that prevent deadenylation without compromising protein expression

ABSTRACTChemical modifications enable preparation of mRNAs with augmented stability and translational activity. In this study, we explored how chemical modifications of 5’,3’-phosphodiester bonds in the mRNA body and polyA tail influence the biological properties of eukaryotic mRNA. To obtain modified and unmodified in vitro transcribed mRNAs, we used ATP and ATP analogues modified at the α-phosphate (containing either O-to-S or O-to-BH3 substitutions) and three different RNA polymerases—SP6, T7 and polyA polymerase. To verify the efficiency of incorporation of ATP analogues in the presence of ATP, we developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for quantitative assessment of modification frequency based on exhaustive degradation of the transcripts to 5’-mononucleotides. The method also estimated the average polyA tail lengths, thereby providing a versatile tool for establishing a structure-biological property relationship for mRNA. We found that mRNAs containing phosphorothioate groups within the polyA tail were substantially less susceptible to degradation by 3’-deadenylase than unmodified mRNA and were efficiently expressed in cultured cells, which makes them useful research tools and potential candidates for future development of mRNA-based therapeutics.

Linear Prediction of Nucleotides in a Genome Sequence

Nucleotides are organic molecules, which are monomer units that form polymers of nucleic acid „deoxyribonucleic acid (DNA)‟ and „ribonucleic acid (RNA)‟. The four nucleotides A, T, G and C get connected by phosphodiester bonds to form strands. Strand formation depends on innumerable factors related to inter and intra cellular parameters and functions. One cannot precisely say that a particular strand gets formed using such and such rules. The infinite possibilities of strand formation cannot be determined experimentally or in the framework of classical genetics. One can alternatively formulate a notion of the “Language of Genomes” wherein one can finitely specify infinite strands. This paper introduces a novel prediction algorithm, which generates possible strands based on available nucleotides statistics.

Structure of Csx1-cOA4 complex reveals the basis of RNA decay in Type III-B CRISPR-Cas

Type III CRISPR-Cas multisubunit complexes cleave ssRNA and ssDNA. These activities promote the generation of cyclic oligoadenylate (cOA), which activates associated CRISPR-Cas RNases from the Csm/Csx families, triggering a massive RNA decay to provide immunity from genetic invaders. Here we present the structure of Sulfolobus islandicus (Sis) Csx1-cOA4 complex revealing the allosteric activation of its RNase activity. SisCsx1 is a hexamer built by a trimer of dimers. Each dimer forms a cOA4 binding site and a ssRNA catalytic pocket. cOA4 undergoes a conformational change upon binding in the second messenger binding site activating ssRNA degradation in the catalytic pockets. Activation is transmitted in an allosteric manner through an intermediate HTH domain, which joins the cOA4 and catalytic sites. The RNase functions in a sequential cooperative fashion, hydrolyzing phosphodiester bonds in 5′-C-C-3′. The degradation of cOA4 by Ring nucleases deactivates SisCsx1, suggesting that this enzyme could be employed in biotechnological applications.

Peroxidase from proso millet exhibits endonuclease-like activity

In this study, the mechanism of DNA cleavage by cationic peroxidase from proso millet (PmPOD) was investigated. PmPOD cleaved supercoiled circular DNA into both nicked circular and linear forms via a cleavage mechanism that resembles those of native endonucleases. Inhibition and ligation studies demonstrated that reactive oxygen species and the ferriprotoporphyrin IX moiety in PmPOD are not involved in PmPOD-mediated DNA cleavage. Similar to other endonucleases, Mg ions considerably enhance the DNA cleavage activity of PmPOD. Further studies suggested that PmPOD can disrupt phosphodiester bonds in DNA and mononucleotides, indicating that it is a phosphatase. The phosphatase activity of PmPOD is higher than that of horseradish peroxidase (HRP), but the peroxidase activity of PmPOD was lower than that of HRP. PmPOD-mediated hydrolytic cleavage of DNA observed in this study is different from those reported for heme proteins. This study provides valuable insights into the distinct mechanisms underlying DNA cleavage by heme proteins.

Cross-linking of DNA Segments by Histone H1 Explains H1 Associated Phenomena of Chromatin Folding

Molecular mechanism for the cooperative and non-cooperative binding of histone H1 to DNA, its rearrangement and exchange between chromatin fibers and its role in the folding of interphase chromatin are proposed in this communication. The mechanism of H1 binding to DNA described here is simple and is based on two established facts; (i) histone H1 can crosslink two DNA segments through salt bridge formation between its positively charged lysine and arginine residues and the negatively charged phosphodiester bonds of the DNA segments, (ii) cations reduce the negative charges on DNA segments and thus decrease the force of repulsion between them.

Can cerium oxide serve as a phosphodiesterase-mimetic nanozyme?

Nanoceria accelerates dramatically not only the dephosphorylation of energetically rich biomolecules such as adenosine triphosphate (ATP), but also the cleavage of highly resistant phosphodiester bonds in 3′,5′-cyclic adenosine monophosphate (cAMP).

Thermococcus Eurythermalis Endonuclease IV Can Cleave Various Apurinic/Apyrimidinic Site Analogues in ssDNA and dsDNA

Endonuclease IV (EndoIV) is a DNA damage-specific endonuclease that mainly hydrolyzes the phosphodiester bond located at 5′ of an apurinic/apyrimidinic (AP) site in DNA. EndoIV also possesses 3′-exonuclease activity for removing 3′-blocking groups and normal nucleotides. Here, we report that Thermococcus eurythermalis EndoIV (TeuendoIV) shows AP endonuclease and 3′-exonuclease activities. The effect of AP site structures, positions and clustered patterns on the activity was characterized. The AP endonuclease activity of TeuendoIV can incise DNA 5′ to various AP site analogues, including the alkane chain Spacer and polyethylene glycol Spacer. However, the short Spacer C2 strongly inhibits the AP endonuclease activity. The kinetic parameters also support its preference to various AP site analogues. In addition, the efficient cleavage at AP sites requires ≥2 normal nucleotides existing at the 5′-terminus. The 3′-exonuclease activity of TeuendoIV can remove one or more consecutive AP sites at the 3′-terminus. Mutations on the residues for substrate recognition show that binding AP site-containing or complementary strand plays a key role for the hydrolysis of phosphodiester bonds. Our results provide a comprehensive biochemical characterization of the cleavage/removal of AP site analogues and some insight for repairing AP sites in hyperthermophile cells.

Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

ABSTRACTDiversity-generating retroelements (DGRs) create unparalleled levels of protein sequence variation through mutagenic retrohoming. Sequence information is transferred from an invariant template region (TR), through an RNA intermediate, to a protein-coding variable region. Selective infidelity at adenines during transfer is a hallmark of DGRs from disparate bacteria, archaea, and microbial viruses. We recapitulated selective infidelityin vitrofor the prototypicalBordetellabacteriophage DGR. A complex of the DGR reverse transcriptase bRT and pentameric accessory variability determinant (Avd) protein along with DGR RNA were necessary and sufficient for synthesis of template-primed, covalently linked RNA-cDNA molecules, as observedin vivo. We identified RNAcDNA molecules to be branched and most plausibly linked through 2′-5′ phosphodiester bonds. Adenine-mutagenesis was intrinsic to the bRT-Avd complex, which displayed unprecedented promiscuity while reverse transcribing adenines of either DGR or non-DGR RNA templates. In contrast, bRT-Avd processivity was strictly dependent on the template, occurring only for the DGR RNA. This restriction was mainly due to a noncoding segment downstream ofTR, which specifically bound Avd and created a privileged site for processive polymerization. Restriction to DGR RNA may protect the host genome from damage. These results define the early steps in a novel pathway for massive sequence diversification.