Biology subjects

Athukoralage, J. S.

Publications and source records attributed to Athukoralage, J. S..

9 recordsLinked to original sources

bioRxiv2026

Autonomous AI agents discover reverse transcriptases with tandem repeat arrays

Metagenomic databases hold vast quantities of unexplored genetic material that can seed tools for biotechnology and medicine, but discovery is limited by our ability to examine sequence data at scale. Here, we show that large language models (LLMs) in an autonomous harness can alleviate this bottleneck and surface novel molecular systems. We deployed Claude Code instances to survey reverse transcriptase (RT) loci across 1.9 billion protein clusters. Among the top candidates were array-associated RTs (ART), a novel family of jumbo-phage RTs coupled with an array of ~200-nucleotide units and a dedicated partner gene. Session transcripts show that Claude identified the ART family by directly examining its DNA sequences and recognizing the atypical repeats, a behavior attributable to specific Mythos 5 internal signals that respond to repeated DNA. ART arrays are highly expressed and appear as discrete units during Staphylococcus phage infection, suggesting an RT system directed by a repertoire of distinct RNAs. These findings indicate that LLMs can autonomously detect anomalies and drive analyses to initiate biological discoveries.

bioinformatics↗

bioRxiv2024

Bridge RNAs direct modular and programmable recombination of target and donor DNA

Genomic rearrangements, encompassing mutational changes in the genome such as insertions, deletions, or inversions, are essential for genetic diversity. These rearrangements are typically orchestrated by enzymes involved in fundamental DNA repair processes such as homologous recombination or in the transposition of foreign genetic material by viruses and mobile genetic elements (MGEs). We report that IS110 insertion sequences, a family of minimal and autonomous MGEs, express a structured non-coding RNA that binds specifically to their encoded recombinase. This bridge RNA contains two internal loops encoding nucleotide stretches that base-pair with the target DNA and donor DNA, which is the IS110 element itself. We demonstrate that the target-binding and donor-binding loops can be independently reprogrammed to direct sequence-specific recombination between two DNA molecules. This modularity enables DNA insertion into genomic target sites as well as programmable DNA excision and inversion. The IS110 bridge system expands the diversity of nucleic acid-guided systems beyond CRISPR and RNA interference, offering a unified mechanism for the three fundamental DNA rearrangements required for genome design.

genetics↗

bioRxiv2020

Tetramerisation of the CRISPR ring nuclease Csx3 facilitates cyclic oligoadenylate cleavage

Type III CRISPR systems detect foreign RNA and activate the cyclase domain of the Cas10 subunit, generating cyclic oligoadenylate (cOA) molecules that act as a second messenger to signal infection, activating nucleases that degrade the nucleic acid of both invader and host. This can lead to dormancy or cell death; to avoid this, cells need a way to remove cOA from the cell once a viral infection has been defeated. Enzymes specialised for this task are known as ring nucleases, but are limited in their distribution. Here, we demonstrate that the widespread CRISPR associated protein Csx3, previously described as an RNA deadenylase, is a ring nuclease that rapidly degrades cyclic tetra-adenylate (cA4). The enzyme has an unusual cooperative reaction mechanism involving an active site that spans the interface between two dimers, sandwiching the cA4 substrate. We propose the name Crn3 (CRISPR associated ring nuclease 3) for the Csx3 family.

biochemistry↗

bioRxiv2020

Fuse to defuse: a self-limiting ribonuclease-ring nuclease fusion for type III CRISPR defence

Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) second messengers in response to viral infection of bacteria and archaea, potentiating an immune response by binding and activating ancillary effector nucleases such as Csx1. As these effectors are not specific for invading nucleic acids, a prolonged activation can result in cell dormancy or death. To avoid this fate, some archaeal species encode a specialised ring nuclease enzyme (Crn1) to degrade cyclic tetra-adenylate (cA4) and deactivate the ancillary nucleases. Some archaeal viruses and bacteriophage encode a potent ring nuclease anti-CRISPR, AcrIII-1, to rapidly degrade cA4 and neutralise immunity. Homologues of this enzyme (named Crn2) exist in type III CRISPR systems but are uncharacterised. Here we describe an unusual fusion between cA4-activated CRISPR ribonuclease (Csx1) and a cA4-degrading ring nuclease (Crn2) from Marinitoga piezophila. The protein has two binding sites that compete for the cA4 ligand, a canonical cA4-activated ribonuclease activity in the Csx1 domain and a potent cA4 ring nuclease activity in the C-terminal Crn2 domain. The activities of the two constituent enzymes in the fusion protein cooperate to ensure a robust but time-limited cOA-activated ribonuclease activity that is finely tuned to cA4 levels as a second messenger of infection.

biochemistry↗

bioRxiv2020

The dynamic interplay of host and viral enzymes in type III CRISPR-mediated cyclic nucleotide signalling

Cyclic nucleotide second messengers are increasingly implicated in prokaryotic anti-viral defence systems. Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) upon detecting foreign RNA, activating ancillary nucleases that can be toxic to cells, necessitating mechanisms to remove cOA in systems that operate via immunity rather than abortive infection. Previously, we demonstrated that the Sulfolobus solfataricus type III-D CRISPR complex generates cyclic tetra-adenylate (cA4), activating the ribonuclease Csx1, and showed that subsequent RNA cleavage and dissociation acts as an "off-switch" for the cyclase activity (Rouillon et al., 2018). Subsequently, we identified the cellular ring nuclease Crn1, which slowly degrades cA4 to reset the system, and demonstrated that viruses can subvert type III CRISPR immunity by means of a potent anti-CRISPR ring nuclease variant. Here, we present a comprehensive analysis of the dynamic interplay between these enzymes, governing cyclic nucleotide levels and infection outcomes in virus-host conflict.

biochemistry↗

bioRxiv2019

A viral ring nuclease anti-CRISPR subverts type III CRISPR immunity

The CRISPR system provides adaptive immunity against mobile genetic elements in bacteria and archaea. On detection of viral RNA, type III CRISPR systems generate a cyclic oligoadenylate (cOA) second messenger1-3, activating defence enzymes and sculpting a powerful antiviral response that can drive viruses to extinction4,5. Cyclic nucleotides are increasingly implicated as playing an important role in host-pathogen interactions6,7. Here, we identify a widespread new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease (AcrIII-1) is the first Acr described for type III CRISPR systems and is widely distributed in archaeal and bacterial viruses, and proviruses. The enzyme uses a novel fold to bind cA4 specifically and utilizes a conserved active site to rapidly cleave the signalling molecule, allowing viruses to neutralise the type III CRISPR defence system. The AcrIII-1 family has a broad host range as it targets cA4 signalling molecules rather than specific CRISPR effector proteins. This study highlights the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.

biochemistry↗

bioRxiv2019

Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence

The CRISPR system provides adaptive immunity against mobile genetic elements (MGE) in prokaryotes. In type III CRISPR systems, an effector complex programmed by CRISPR RNA detects invading RNA, triggering a multi-layered defence that includes target RNA cleavage, licencing of an HD DNA nuclease domain and synthesis of cyclic oligoadenylate (cOA) molecules. cOA activates the Csx1/Csm6 family of effectors, which degrade RNA non-specifically to enhance immunity. Type III systems are found in diverse archaea and bacteria, including the human pathogen Mycobacterium tuberculosis. Here, we report a comprehensive analysis of the in vitro and in vivo activities of the type III-A M. tuberculosis CRISPR system. We demonstrate that immunity against MGE is achieved predominantly via a cyclic hexa-adenylate (cA6) signalling pathway and the ribonuclease Csm6, rather than through DNA cleavage by the HD domain. Furthermore, we show that the mechanism can be reprogrammed to operate as a cyclic tetra-adenylate (cA4) system by replacing the effector protein. These observations demonstrate that M. tuberculosis has a fully-functioning CRISPR interference system that generates a range of cyclic and linear oligonucleotides of known and unknown functions, potentiating fundamental and applied studies.

biochemistry↗

bioRxiv2019

A type III CRISPR ancillary ribonuclease degrades its cyclic oligoadenylate activator

Cyclic oligoadenylate (cOA) secondary messengers are generated by type III CRISPR systems in response to viral infection. cOA allosterically activates the CRISPR ancillary ribonucleases Csx1/Csm6, which degrade RNA non-specifically using a HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) active site. This provides effective immunity, but can also lead to growth arrest in infected cells, necessitating a means to deactivate the ribonuclease once viral infection has been cleared. In the crenarchaea, dedicated ring nucleases degrade cA4 (cOA consisting of 4 AMP units), but the equivalent enzyme has not been identified in bacteria. We demonstrate that, in Thermus thermophilus HB8, the uncharacterised protein TTHB144 is a cA4-activated HEPN ribonuclease that also degrades its activator. TTHB144 binds and degrades cA4 at an N-terminal CARF (CRISPR Associated Rossman Fold) domain. The two activities can be separated by site-directed mutagenesis. TTHB144 is thus the first example of a self-limiting CRISPR ribonuclease.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC=\"FIGDIR/small/582114_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (21K):\norg.highwire.dtl.DTLVardef@d4ac46org.highwire.dtl.DTLVardef@7a67b0org.highwire.dtl.DTLVardef@2d8c7aorg.highwire.dtl.DTLVardef@61b12a_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

biochemistry↗

bioRxiv2018

Ring nucleases deactivate Type III CRISPR ribonucleases by degrading cyclic oligoadenylate

The CRISPR-Cas system provides adaptive immunity against mobile genetic elements in prokaryotes, utilising small CRISPR RNAs which direct effector complexes to degrade invading entities. Type III effector complexes were recently demonstrated to synthesise a novel second messenger, cyclic oligoadenylate (cOA), on binding target RNA. cOA in turn binds to and activates a range of downstream effector proteins including ribonucleases (Csm6/Csx1) and transcription factors via a CARF (CRISPR associated Rossman Fold) domain, inducing an antiviral state in the cell that is important for immunity. The mechanism of the \"off-switch\" that resets the system is not understood. Here, we report the identification of the nuclease that degrades these cOA ring molecules. The \"Ring nuclease\" is itself a CARF family protein with a metal independent mechanism, which cleaves cOA4 rings to generate linear di-adenylate species and switches off the antiviral state. The identification of Ring nucleases adds an important insight to the CRISPR-Cas system.

biochemistry↗