Rift Valley fever (RVF) virus is widespread worldwide and poses a severe threat to human life and property. RVF viral polymerase plays a vital role in the replication and transcription of the virus. Here, we describe how to express and purify this polymerase and perform tests for its in vitro activity assays.

INTS11 and CPSF73 are metal-dependent endonucleases for Integrator and pre-mRNA 3\'-end processing, respectively. Here, we show that the INTS11 binding partner BRAT1/CG7044, a factor important for neuronal fitness, stabilizes INTS11 in the cytoplasm and is required for Integrator function in the nucleus. Loss of BRAT1 in neural organoids leads to transcriptomic disruption and precocious expression of neurogenesis-driving transcription factors. The structures of the human INTS9-INTS11-BRAT1 and Drosophila dIntS11-CG7044 complexes reveal that the conserved C terminus of BRAT1/CG7044 is captured in the active site of INTS11, with a cysteine residue directly coordinating the metal ions. Inspired by these observations, we find that UBE3D is a binding partner for CPSF73, and UBE3D likely also uses a conserved cysteine residue to directly coordinate the active site metal ions. Our studies have revealed binding partners for INTS11 and CPSF73 that behave like cytoplasmic chaperones with a conserved impact on the nuclear functions of these enzymes.

Here, we report a novel endonuclease and N6-adenine DNA methyltransferase (m6A methyltransferase) in the Ureaplasma parvum SV3F4 strain. Our previous study found that the SV3F4 strain carries 17 unique genes, which are not encoded in the two previously reported U. parvum serovar 3 strain, OMC-P162 and ATCC 700970. Of these 17 unique genes, UP3_c0261 and UP3_c0262, were originally annotated as encoding hypothetical proteins. Comparative genomics analyses more recently indicated they encode a Type II restriction endonuclease and an m6A methyltransferase, respectively. The UP3_c0261 and UP3_c0262 genes were individually expressed and purified in Escherichia coli. The UP3_c0261 recombinant protein showed endonuclease activity on the pT7Blue vector, recognizing and cleaving a GTNAC motif, resulting in a 5 base 5\' extension. The UP3_c0261 protein digested a polymerase chain reaction (PCR) product harboring the GTNAC motif. The endonuclease UP3_c0261 was designated as UpaF4I. Treatment of the PCR product with the recombinant protein UP3_c0262 completely blocked the restriction enzyme activity of UpaF4I. Analysis of the treated PCR product harboring a modified nucleotide by UP3_c0262 with HPLC-MS/MS and MS/MS showed that UP3_c0262 was an m6A methyltransferase containing a methylated A residue in both DNA strands of the GTNAC motif. Whole genome methylation analysis of SV3F4 showed that 99.9 % of the GTNAC motif was m6A modified. These results suggest the UP3_c0261 and UP3_c0262 genes may act as a novel Type II restriction-modification system in the Ureaplasma SV3F4 strain.

This perspective underscores the rising challenge posed by emerging diseases against the backdrop of modern advancements in global public health understanding. It particularly highlights the emergence of the Oropouche virus (OROV) as a significant global threat, detailing its transmission dynamics, symptoms, and epidemiological impact, with a focus on its historical and current manifestations. It further delves into the molecular aspects of OROV, elucidating its unique characteristics, lack of structural similarity with other arboviruses, and the limited progress in medicinal chemistry research. Still, it highlights notable studies on potential antiviral agents and the challenges in drug development, emphasizing the need for innovative approaches such as structure-based drug design (SBDD) and drug repurposing. Finally, it concludes with a call to action, urging increased attention and research focus on OROV to prevent potential future pandemics fueled by viral mutations.

We report the biochemical, structural, and functional characterization of the protein coded by gene PA4880 in the P. aeruginosa PAO1 genome. The PA4880 gene had been annotated as coding a probable bacterioferritin. Our structural work shows that the product of gene PA4880 is a protein that adopts the Dps subunit fold, which oligomerizes into a 12-mer quaternary structure. Unlike Dps, however, the ferroxidase di-iron centers and iron coordinating ligands are buried within each subunit, in a manner identical to that observed in the ferroxidase center of P. aeruginosa bacterioferritin. Since these structural characteristics correspond to Dps-like proteins, we term the protein as P. aeruginosa Dps-like, or Pa DpsL. The ferroxidase centers in Pa DpsL catalyze the oxidation of Fe2+ utilizing O2 or H2O2 as oxidant, and the resultant Fe3+ is compartmentalized in the interior cavity. Interestingly, incubating Pa DpsL with plasmid DNA results in efficient nicking of the DNA and at higher concentrations of Pa DpsL the DNA is linearized and eventually degraded. The nickase and endonuclease activities suggest that Pa DpsL, in addition to participating in the defense of P. aeruginosa cells against iron-induced toxicity, may also participate in the innate immune mechanisms consisting of restriction endonucleases and cognate methyl transferases.

Viral infection triggers several double-stranded RNA (dsRNA) sensors that lead to changes in gene expression in the cell. One of these sensors activates an endonuclease, ribonuclease L (RNase L), that cleaves single-stranded RNA. However, how the resultant widespread RNA fragmentation affects gene expression is not fully understood. Here, we show that this fragmentation induces the ribotoxic stress response via ZAKα, potentially through stalled ribosomes and/or ribosome collisions. The p38 and JNK pathways that are activated as part of this response promote outcomes that inhibit the virus, such as programmed cell death. We also show that RNase L limits the translation of stress-responsive genes. Intriguingly, we found that the activity of the generic endonuclease, RNase A, recapitulates many of the same molecular phenotypes as activated RNase L, demonstrating how widespread RNA cleavage can evoke an antiviral program.

The DNA mismatch repair (MMR) system promotes genome stability and protects humans from certain types of cancer. Its primary function is the correction of DNA polymerase errors. MutLα is an important eukaryotic MMR factor. We have examined the contributions of MutLα to maintaining genome stability. We show here that loss of MutLα in yeast increases the genome-wide mutation rate by ∼130-fold and generates a genome-wide mutation spectrum that consists of small indels and base substitutions. We also show that loss of yeast MutLα leads to error-prone MMR that produces T > C base substitutions in 5\'-ATA-3\' sequences. In agreement with this finding, our examination of human whole-genome DNA sequencing data has revealed that loss of MutLα in induced pluripotent stem cells triggers error-prone MMR that leads to the formation of T > C mutations in 5\'-NTN-3\' sequences. Our further analysis has shown that MutLα-independent MMR plays a role in suppressing base substitutions in N3 homopolymeric runs. In addition, we describe that MutLα preferentially protects noncoding DNA from mutations. Our study defines the contributions of MutLα-dependent and independent mechanisms to genome-wide MMR.

Thermophilic Argonaute proteins (Agos) have been shown to utilize small DNA guides for cleaving complementary DNA in vitro, which shows great potential for nucleic acid detection. In this study, we explored mesophilic Agos for the detection of small molecule by cooperating with allosteric transcription factors (aTFs). Two Agos from mesophilic bacteria, Paenibacillus borealis (PbAgo) and Brevibacillus laterosporus (BlAgo), showed nuclease activity for single-stranded DNA at moderate temperatures (37 °C) by using 5\'-phosphorylated and 5\'-hydroxylated DNA guides. Both Agos perform programmable cleavage of double-stranded DNA, especially in AT-rich regions of plasmid. Furthermore, we developed a simple and low-cost p-hydroxybenzoic acid detection method based on DNA-guided DNA cleavage of Agos and the allosteric effect of HosA, which expands the potential application of small molecule detection by Agos.

BACKGROUND: Prokaryotic Argonaute (pAgo) proteins are well-known oligonucleotide-directed endonucleases, which contain a conserved PIWI domain required for endonuclease activity. Distantly related to pAgos, PIWI-RE family, which is defined as PIWI with conserved R and E residues, has been suggested to exhibit divergent activities. The distinctive biochemical properties and physiological functions of PIWI-RE family members need to be elucidated to explore their applications in gene editing.
RESULTS: Here, we describe the catalytic performance and cellular functions of a PIWI-RE family protein from Pseudomonas stutzeri (PsPIWI-RE). Structural modelling suggests that the protein possesses a PIWI structure similar to that of pAgo, but with different PAZ-like and N-terminal domains. Unlike previously reported pAgos, recombinant PsPIWI-RE acts as an RNA-guided DNA nuclease, as well as a DNA-guided RNA nuclease. It cleaves single-stranded DNA at temperatures ranging from 20 to 65 °C, with an optimum temperature of 45 °C. Mutation at D525 or D610 significantly reduced its endonuclease activity, confirming that both residues are key for catalysis. Comparing with wild-type, mutant with PIWI-RE knockout is more sensitive to ciprofloxacin as DNA replication inhibitor, suggesting PIWI-RE may potentially be involved in DNA replication.
CONCLUSIONS: Our study provides the first insights into the programmable nuclease activity and biological function of the unknown PIWI-RE family of proteins, emphasizing their important role in vivo and potential application in genomic DNA modification.

Restriction modification (RM) systems are one of the ubiquitous yet primitive defense responses employed by bacteria and archaea with the primary role of safeguarding themselves against invading bacteriophages. Protection of the host occurs by the cleavage of the invading foreign DNA via restriction endonucleases with concomitant methylation of host DNA with the aid of a methyltransferase counterpart. RM systems have been extensively studied in bacteria, however, in the case of archaea there are limited reports of RM enzymes that are investigated to date owing to their inhospitable growth demands. This review aims to broaden the knowledge about what is known about the diversity of RM systems in archaea and encapsulate the current knowledge on restriction and modification enzymes characterized in archaea so far and the role of RM systems in the milieu of archaeal biology.