Biology subjects

Lampe, G.

Publications and source records attributed to Lampe, G..

3 recordsLinked to original sources

bioRxiv2026

Mechanism of tandem-repeat DNA synthesis by an antiviral reverse transcriptase

Defense-associated reverse transcriptases (DRTs) employ DNA synthesis to protect bacteria against phage infection1,2. We previously showed that DRT10, a tripartite system comprising an RT, a noncoding RNA (ncRNA), and a SLATT effector protein, catalyzes protein-primed, tandem-repeat DNA synthesis in a mechanism strikingly analogous to eukaryotic telomerase3. However, the structural basis by which the RT-ncRNA complex directs repeat addition processivity and controls repeat length remains unknown. Here we present cryo-EM reconstructions of two evolutionarily diverse DRT10 RT-ncRNA systems that reveal an unanticipated 2:1 architecture, wherein two RT monomers bind opposite sides of a single, pseudo-symmetric ncRNA. Biochemical experiments demonstrate that each RT monomer reverse transcribes the template encoded on its respective side of the ncRNA, but only one generates the long repetitive product, with the template sequence defined by the distance between two flanking stem-loop anchors. Together with earlier studies of DRT2, DRT3, and DRT94-6, our findings identify a conserved mechanistic logic underlying ncRNA-templated tandem-repeat synthesis across Class 2 UG antiviral systems, despite vastly different architectural solutions.

microbiology↗

bioRxiv2026

Structural basis of RNA-guided DNA integration by type I CRISPR-associated transposases

CRISPR-associated transposases (CASTs) achieve site-specific DNA integration by coupling the RNA-guided targeting action of a nuclease-deficient CRISPR-Cas system with the assembly of a Tn7-like transpososome complex1,2. Understanding the detailed mechanisms of this elaborate process is paramount to engineering CAST systems into programmable genetic tools3-6. The type I-F Pseudoalteromonas CAST (PseCAST) displays the highest activity in mammalian cells to date7 and has been the subject of extensive directed evolution8, but efforts to rationally engineer further improvements have been hampered by critical gaps in our understanding of transpososome assembly and activation9. Here we use cryo-EM structural analysis, validated by DNA transposition assays, to visualize the PseCAST system in a series of functional states that define the stepwise mechanism of RNA-guided DNA integration. The structure of a target DNA-bound Cascade-TniQ-TnsC complex reveals that conformational changes induced by R-loop formation are coupled to target DNA stabilization and TnsC heptamerization, which in turn recruits the TnsAB transposase via conserved interactions with its C-terminal tail. Finally, the structure of the 1.2 MDa PseCAST transpososome holocomplex reveals specific TnsC-TnsB and TnsB-target DNA interactions that drive allosteric remodelling of the TnsB catalytic site to activate donor DNA integration. Together, these findings establish a unified structural and mechanistic blueprint for RNA-guided DNA integration and lay the foundation for engineering next-generation DNA insertion systems for genome editing applications.

molecular biology↗

bioRxiv2024

TGF-β induces an atypical EMT to evade immune mechanosurveillance in lung adenocarcinoma dormant metastasis

The heterogeneity of epithelial-to-mesenchymal transition (EMT) programs is manifest in the diverse EMT-like phenotypes occurring during tumor progression. However, little is known about the mechanistic basis and functional role of specific forms of EMT in cancer. Here we address this question in lung adenocarcinoma (LUAD) cells that enter a dormancy period in response to TGF-{beta} upon disseminating to distant sites. LUAD cells with the capacity to enter dormancy are characterized by expression of SOX2 and NKX2-1 primitive progenitor markers. In these cells, TGF-{beta} induces growth inhibition accompanied by a full EMT response that subsequently transitions into an atypical mesenchymal state of round morphology and lacking actin stress fibers. TGF-{beta} induces this transition by driving the expression of the actin-depolymerizing factor gelsolin, which changes a migratory, stress fiber-rich mesenchymal phenotype into a cortical actin-rich, spheroidal state. This transition lowers the biomechanical stiffness of metastatic progenitors, protecting them from killing by mechanosensitive cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Inhibiting this actin depolymerization process clears tissues of dormant metastatic cells. Thus, LUAD primitive progenitors undergo an atypical EMT as part of a strategy to evade immune-mediated elimination during dormancy. Our results provide a mechanistic basis and functional role of this atypical EMT response of LUAD metastatic progenitors and further illuminate the role of TGF-{beta} as a crucial driver of immune evasive metastatic dormancy.

cancer biology↗