Biology subjects

Loeff, L.

Publications and source records attributed to Loeff, L..

7 recordsLinked to original sources

bioRxiv2026

Processing of forked DNA activates a helicase-nuclease immune system

Innate immune systems detect molecular signatures of infection to initiate antiviral defence1-3, yet the identity of pathogen-associated signals that distinguish phage from host nucleic acids remains incompletely understood. While recent work has shown that nucleic acid structures can act as triggers for bacterial defense systems4-7, how these structural signals are coupled with immune activation remain unclear. Here we show that forked DNA structures activate a helicase-nuclease immune complex in type III Druantia through a processing-dependent mechanism. Using cryo-electron microscopy and biochemical reconstitution, we find that the exonuclease DruH processes 3' DNA termini to generate 5' overhangs that recruit and activate the helicase-nuclease DruE at duplex-single-stranded DNA junctions. Structural analysis of the DruE-DruH complex reveals how substrate-dependent assembly remodels an autoinhibited helicase dimer into an active DNA degradation complex. Functional assays demonstrate that coordinated nuclease and helicase activities enable efficient degradation of forked DNA substrates and mediate phage defense without detectable host toxicity. Together, our findings define a mechanism in which enzymatic processing of replication-associated DNA structures licenses immune activation, providing a framework for how nucleic acid architecture is coupled to effector activation in bacterial immunity.

microbiology↗

bioRxiv2025

Bacterial Schlafens mediate anti-phage defense

Human Schlafen proteins restrict viral replication by cleaving tRNA, thereby suppressing protein synthesis. Although the ribonuclease domain of Schlafen proteins is conserved across all domains of life, its function in prokaryotes has remained unclear. Here, we show that prokaryotic Schlafen nucleases (pSlfns) are widespread antiviral effectors that protect bacteria from phages. These nucleases are fused to diverse protein domains that sense phage infection. We focus on a system where Schlafen nuclease is fused to a previously unknown immunoglobulin-like sensor domain and demonstrate that it recognizes T5-like phage tail assembly chaperones and cleaves both bacterial and viral tRNA, triggering abortive infection. Our findings redefine Schlafens as an ancient, mechanistically conserved family of immune effectors, revealing the deep evolutionary origin of tRNA-targeting antiviral immunity in humans.

molecular biology↗

bioRxiv2024

Molecular mechanism of plasmid elimination by the DdmDE defense system

Seventh pandemic Vibrio cholerae strains contain two hallmark pathogenicity islands that encode the DNA defense modules DdmABC and DdmDE. Here we use cryo-EM to reveal the mechanistic basis for plasmid defense by DdmDE. A cryo-EM structure of the DdmD helicase-nuclease reveals that it adopts an auto-inhibited dimeric architecture. The prokaryotic Argonaute protein DdmE uses a DNA guide to target plasmid DNA. A structure of the DdmDE complex, validated by in vivo mutational studies, shows that DNA binding by DdmE triggers disassembly of the DdmD dimer and loading of monomeric DdmD onto the non-target DNA strand. Finally, in vitro studies reveal that DdmD translocates in the 5 to 3 direction, while partially degrading the plasmid DNA. These findings provide critical insights into the architecture and mechanism of DdmDE systems in plasmid elimination.

microbiology↗

bioRxiv2023

DNA end sensing and cleavage by the Shedu anti-phage defense system

The detection of molecular patterns associated with invading pathogens is a hallmark of innate immune systems. Prokaryotes deploy sophisticated host-defense mechanisms in innate anti-phage immunity. Shedu is a single-component defense system comprising a putative nuclease SduA. Here we report cryo-EM structures of apo- and dsDNA-bound tetrameric SduA assemblies, revealing that the N-terminal domains of SduA form a clamp that recognizes free DNA ends. End binding positions the DNA over the PD/ExK nuclease domain, resulting in dsDNA nicking at a fixed distance from the 5 end. The end-directed DNA nicking activity of Shedu prevents propagation of linear DNA in vivo. Finally, we show that phages escape Shedu immunity by suppressing their recombination-dependent DNA replication pathway. Taken together, these results define the antiviral mechanism of Shedu systems, underlining the paradigm that recognition of pathogen-specific nucleic acid structures is a conserved feature of innate immunity across all domains of life.

biochemistry↗

bioRxiv2023

Mechanistic basis for oxidative stress protection of the human tRNA ligase complex by the oxidoreductase PYROXD1

RTCB is the catalytic subunit of the metazoan tRNA ligase complex (tRNA-LC) that plays essential roles in tRNA biogenesis and unfolded protein response. The catalytic center of RTCB contains a conserved cysteine that is susceptible to metal ion-induced oxidative inactivation. The flavin-containing oxidoreductase PYROXD1 preserves the activity of mammalian tRNA-LC in a NAD(P)H-dependent manner, but its protective mechanism remains elusive. Here we report a cryo-EM structure of human RTCB in complex with PYROXD1, revealing that PYROXD1 directly interacts with the catalytic center of RTCB through its C-terminal tail. NAD(P)H binding and FAD reduction allosterically control PYROXD1 activity and RTCB recruitment and PYROXD1, while PYROXD1 reoxidation enables timed release of RTCB. PYROXD1 interaction is mutually exclusive with Archease-mediated RTCB guanylylation, and guanylylated RTCB is intrinsically protected from oxidative inactivation. Together, these findings provide a mechanistic framework for the protective function of PYROXD1 that maintains the activity of tRNA-LC under aerobic conditions.

biochemistry↗

bioRxiv2019

Selective Prespacer Processing Ensures Precise CRISPR-Cas Adaptation

CRISPR-Cas immunity protects prokaryotes against foreign genetic elements. CRISPR-Cas uses the highly conserved Cas1-Cas2 complex to establish inheritable memory (spacers). It remains elusive how Cas1-Cas2 acquires spacers from cellular DNA fragments (prespacers) and how it integrates them into the CRISPR array in the correct orientation. By using the high spatiotemporal resolution of single-molecule fluorescence, we reveal that Cas1-Cas2 obtains prespacers in various forms including single-stranded DNA and partial duplexes by selecting them in the DNA-length and PAM-dependent manner. Furthermore, we identify DnaQ exonucleases as enzymes that can mature the Cas1-Cas2-loaded precursor prespacers into an integration-competent size. Cas1-Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and subsequent spacer integration in the correct orientation. This kinetic coordination in prespacer selection and PAM trimming provides comprehensive understanding of the mechanisms that underlie the integration of functional spacers in the CRISPR array.

biophysics↗

bioRxiv2017

Repetitive DNA reeling by the Cascade-Cas3 complex in nucleotide unwinding steps

CRISPR-Cas loci provide an RNA-guided adaptive immune system against invading genetic elements. Interference in type I systems relies on the RNA-guided surveillance complex Cascade for target DNA recognition and the trans-acting Cas3 helicase/nuclease protein for target degradation. Even though the biochemistry of CRISPR interference has been largely covered, the biophysics of DNA unwinding and coupling of the helicase and nuclease domains of Cas3 remains elusive. Here we employed single-molecule FRET to probe the helicase activity with a high spatiotemporal resolution. We show that Cas3 remains tightly associated with the target-bound Cascade complex while reeling in the target DNA using a spring-loaded mechanism. This spring-loaded reeling occurs in distinct bursts of three base pairs, that each underlie three successive 1-nt unwinding events. Reeling is highly repetitive, compensating for inefficient nicking activity of the nuclease domain. Our study reveals that the discontinuous helicase properties of Cas3 and its tight interaction with Cascade ensure well controlled degradation of target DNA only.

biophysics↗