4G cloning: rapid gene assembly for expression of multisubunit protein complexes in diverse hosts DOI Creative Commons
Michael J. Taschner,

Joe Bradley Dickinson,

Florian Roisné-Hamelin

et al.

Life Science Alliance, Journal Year: 2024, Volume and Issue: 8(1), P. e202402899 - e202402899

Published: Dec. 2, 2024

Multisubunit protein complexes are central to many cellular processes, and studying their activities structures in vitro requires reconstitution via recombinant expression purification. Obtaining targets at sufficient purity scale typically involves screening several variants hosts. Existing cloning strategies enable co-expression but often time-consuming, labor-intensive, host-specific, or involve error-prone steps. We present a novel vector set assembly strategy overcome these limitations, enabling construct generation for multisubunit single step. This modular system can be extended additional hosts include new tags. demonstrate its utility by constructing vectors structural maintenance of chromosomes various hosts, streamlining workflows, improving productivity.

Language: Английский

An extrinsic motor directs chromatin loop formation by cohesin DOI Creative Commons
Thomas M Guérin, Christopher Barrington, Georgii Pobegalov

et al.

The EMBO Journal, Journal Year: 2024, Volume and Issue: 43(19), P. 4173 - 4196

Published: Aug. 19, 2024

Language: Английский

Citations

9
Structure and Activation Mechanism of a Lamassu Phage Defence System DOI Creative Commons
Yan Li, David W. Adams, Hon Wing Liu

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: March 15, 2025

Summary Lamassu is a diverse family of defence systems that protect bacteria, including pandemic strains Vibrio cholerae , against phage infection. They target essential cellular processes, aborting infection and preventing propagation by terminating the infected host. The mechanisms which efectors are activated when needed otherwise suppressed unknown. Here, we present structures system from Salmonella enterica . We show an oligomerization domain nuclease efector, LmuA, sequestered two tightly-folded SMC-like LmuB protomers LmuC. Upon activation, liberated LmuA proteins assemble into cyclic homo-tetramer, in four domains brought proximity to create active site capable cleaving DNA. propose tetramer formation likely one-way switch establishes threshold limit potential spontaneous activation cell death. Our findings reveal mechanism defence, involving liberation immune efectors, shed light on how balance potent responses with self-preservation.

Language: Английский

Citations

1
Loop extrusion-mediated plasmid DNA cleavage by the bacterial SMC Wadjet complex DOI
Biswajit Pradhan, Amar Deep,

Jessica König

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Feb. 17, 2024

Structural maintenance of chromosomes (SMC) protein complexes play pivotal roles in genome organization and across all domains life. In prokaryotes, SMC family Wadjet structurally resemble the widespread MukBEF genome-organizing but serve a defensive role by inhibiting plasmid transformation. We previously showed that specifically cleaves circular DNA; however, molecular mechanism underlying DNA substrate recognition remains unclear. Here, we use vitro single-molecule imaging to directly visualize loop extrusion cleavage Wadjet. find is symmetric extruder simultaneously reels from both sides growing this activity requires dimeric JetABC supercomplex containing two dimers JetC motor subunit. On surface-anchored DNAs, extrudes full length 44 kilobase pair plasmid, stalls, then DNA. Our findings reveal specific elimination plasmids Wadjet, establish as an evolutionarily conserved among kingdoms

Language: Английский

Citations

6
Loop-extrusion-mediated plasmid DNA cleavage by the bacterial SMC Wadjet complex DOI Creative Commons
Biswajit Pradhan, Amar Deep,

Jessica König

et al.

Molecular Cell, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 1, 2024

Structural maintenance of chromosomes (SMC) complexes play pivotal roles in genome organization and across all domains life. In prokaryotes, SMC-family Wadjet structurally resemble the widespread MukBEF but serve a defensive role by inhibiting plasmid transformation. We previously showed that specifically cleaves DNA; however, molecular mechanism underlying recognition remains unclear. Here, we use vitro single-molecule imaging to directly visualize DNA loop extrusion cleavage Wadjet. find is symmetric extruder simultaneously reels from both sides this activity requires dimeric JetABC supercomplex. On surface-anchored DNAs, extrudes full length 44-kb-pair plasmid, stalls, DNA. Our findings reveal specific elimination plasmids establish as an evolutionarily conserved among SMC kingdoms

Language: Английский

Citations

6
Nucleic acid recognition during prokaryotic immunity DOI Creative Commons
Christian F. Baca, Luciano A. Marraffini

Molecular Cell, Journal Year: 2025, Volume and Issue: 85(2), P. 309 - 322

Published: Jan. 1, 2025

Language: Английский

Citations

0
Genome folding by cohesion DOI
Shu‐Tao Qi, Zhubing Shi, Hongtao Yu

et al.

Current Opinion in Genetics & Development, Journal Year: 2025, Volume and Issue: 91, P. 102310 - 102310

Published: Jan. 18, 2025

Language: Английский

Citations

0
A unified model for cohesin function in sisterchromatid cohesion and chromatin loop formation DOI Creative Commons
Frank Uhlmann

Molecular Cell, Journal Year: 2025, Volume and Issue: 85(6), P. 1058 - 1071

Published: March 1, 2025

The ring-shaped cohesin complex topologically entraps two DNAs to establish sister chromatid cohesion. Cohesin also shapes the interphase chromatin landscape by forming DNA loops, which it is thought achieve using an in vitro-observed loop extrusion mechanism. However, recent studies revealed that loop-extrusion-deficient retains its ability form suggesting a divergence of vitro and vivo formation. Instead extrusion, we examine whether forms loops mechanism akin cohesion establishment: sequential topological capture DNAs. We explore similarities differences between "loop capture" extrusion" model, how they compare at explaining experimental observations, future approaches can delineate their possible respective contributions. extend our DNA-DNA model for function related structural maintenance chromosomes (SMC) family members, condensin, Smc5-Smc6 complex, bacterial SMC complexes.

Language: Английский

Citations

0
Structural basis of antiphage defense by an ATPase-associated reverse transcriptase DOI Creative Commons
Jerrin Thomas George, Nathaniel Burman, Royce A. Wilkinson

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: March 26, 2025

Reverse transcriptases (RTs) have well-established roles in the replication and spread of retroviruses retrotransposons. However, recent evidence suggests that RTs been conscripted by cells for diverse antiviral defense. Here we determine structures a type I-A retron, which explain how RNA, DNA, RT, HNH-nuclease four molecules an SMC-family ATPase assemble into 364 kDa complex provides phage We show phage-encoded nucleases trigger degradation retron-associated leading to disassembly retron activation HNH nuclease. The nuclease cleaves tRNA Ser , stalling protein synthesis arresting viral replication. Taken together, these data reveal paradoxical perpetuation elimination genetic parasites.

Language: Английский

Citations

0
Mechanism of DNA capture by the MukBEF SMC complex and its inhibition by a viral DNA mimic DOI Creative Commons
Frank Bürmann, Bryony Clifton,

Sophie Koekemoer

et al.

Cell, Journal Year: 2025, Volume and Issue: unknown

Published: March 1, 2025

Ring-like structural maintenance of chromosome (SMC) complexes are crucial for genome organization and operate through mechanisms DNA entrapment loop extrusion. Here, we explore the loading process bacterial SMC complex MukBEF. Using cryoelectron microscopy (cryo-EM), demonstrate that ATP binding opens one MukBEF's three potential entry gates, exposing a capture site positions at open neck gate. We discover gp5.9 protein bacteriophage T7 blocks this by mimicry, thereby preventing inactivating propose comprehensive unidirectional mechanism in which is first captured complex's periphery then ingested gate, powered single cycle hydrolysis. These findings illuminate fundamental aspect how ubiquitous organizers primed can be disrupted viruses.

Language: Английский

Citations

0
4G cloning: rapid gene assembly for expression of multisubunit protein complexes in diverse hosts DOI Creative Commons
Michael J. Taschner,

Joe Bradley Dickinson,

Florian Roisné-Hamelin

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: June 17, 2024

Abstract Multi-subunit protein complexes are at the heart of many cellular processes, and studying their biochemical activities structures in vitro requires reconstitution by recombinant expression purification. Obtaining targets sufficient purity scale typically screening several variants hosts. Existing cloning strategies allow to produce constructs for co-expression proteins, but often time-consuming, labour-intensive, host-specific, or involving error-prone assembly steps. Here we present a unique set vectors together with novel strategy designed overcome these limitations. It allows generation multi-subunit various hosts single step. Its modular nature system be easily extended target additional include new tags regulatory sequences. As proof principle, parallel construction Structural Maintenance Chromosomes (SMC) complexes, allowing us devise production bacteria, insect cells, human respectively. This work will help laboratories working on streamline workflow, increase productivity improve quality purified material.

Language: Английский

Citations

1