Periodic Reporting for period 1 - LoopSMC (Mechanism, Regulation and Functions of DNA Loop Extrusion by SMC complexes)
Período documentado: 2023-04-01 hasta 2025-09-30
Life and evolution of organisms relies on the maintenance, integration, propagation, and readout of genetic information. This information is stored in chromosomes that have a specific three-dimensional structure, a condensed yet accessible form of DNA that is dynamically folded during the lifespan of cells. How DNA is folded within chromosomes has however remained a mystery. It has been proposed that this is achieved by a process of loop extrusion in which SMC (Structural Maintenance of Chromosomes) complexes that are multi-subunit ATPases present in all kingdoms of life – including condensin and cohesin – reel DNA into loops, thereby organizing genomic DNA into higher-order structures. Recent in vitro single-molecule studies, stimulated by our initial discovery on condensin, provided direct evidences that both condensin and cohesin can indeed generate chromatin loops by
extrusion. However, the most fundamental questions relating to this process remain unanswered: What is the molecular mechanism of loop extrusion? How is this process regulated? What are the functional roles of SMC-mediated loop extrusion beyond condensation? To address these questions, we will synergistically combine our single-molecule loop extrusion assay with correlative light and electron tomography and force spectroscopy to reveal both dynamic and structural aspects of loop extrusion and SMC proteins. Specifically, we will resolve how SMC complexes function as molecular ‘motors’, how regulatory factors modulate the kinetics of loop extrusion, and how loop extrusion impacts cellular functions like chromosome segregation and gene recombination, all at the single molecule level. In the long term, our findings will provide vital insights into the basic packaging structure of the genome which directly governs its biological function.
extrusion. However, the most fundamental questions relating to this process remain unanswered: What is the molecular mechanism of loop extrusion? How is this process regulated? What are the functional roles of SMC-mediated loop extrusion beyond condensation? To address these questions, we will synergistically combine our single-molecule loop extrusion assay with correlative light and electron tomography and force spectroscopy to reveal both dynamic and structural aspects of loop extrusion and SMC proteins. Specifically, we will resolve how SMC complexes function as molecular ‘motors’, how regulatory factors modulate the kinetics of loop extrusion, and how loop extrusion impacts cellular functions like chromosome segregation and gene recombination, all at the single molecule level. In the long term, our findings will provide vital insights into the basic packaging structure of the genome which directly governs its biological function.
The LoopSMC project aimed to elucidate the molecular mechanisms, regulation and biological functions of SMC complexes in genome organization, focusing on loop extrusion as a central activity. In work package 1, we investigated the motor mechanisms of the eukaryotic Smc5/6 complex, building on our initial discovery that Smc5/6 extrudes DNA loops as dimers while individual complexes translocate unidirectionally, and that the Nse5/6 sub-complex modulates this activity (Pradhan et al., Nature, 2023). To uncover the structural basis of these mechanisms, we employed cryo-electron microscopy (cryo-EM) to resolve multiple functional states of Smc5/6—including Nse5/6-bound apo- and ATP-bound forms, as well as apo-, DNA-bound, and DNA–ATP-bound states—at resolutions ranging from ~2–6 Å (Wong et al., in preparation). These structures reveal previously unobserved conformational intermediates across the ATPase cycle and provide detailed mechanistic insight into the translocation and loop extrusion process. To validate the functional relevance of these conformations, we are using a combination of targeted mutagenesis at DNA and Nse5/6 binding interfaces, site-specific crosslinking to enforce or restrict conformational transitions, and downstream single-molecule fluorescence resonance energy transfer (FRET), atomic force microscopy (AFM), and biochemical assays to test their impact on Smc5/6 activity.
In work package 2, we aimed to investigate the regulatory mechanisms of SMC complexes. Our focus shifted from the well-characterized cohesin complex to the less-explored condensin I and II, aiming to understand their regulation by co-factors. For condensin I, we demonstrated that chromokinesin KIF4A regulates loop extrusion by binding to the HAWK subunit NCAPG in its C-terminal tail (Cutts et al., EMBO J., 2024). This interaction activates condensin I’s ATPase and loop extrusion activities, and notably, KIF4A alone is sufficient to trigger this activation. Extending these studies to condensin II, which is nuclear during interphase and activated during mitosis, we are investigating its regulation by candidate factors including the inhibitor MCPH1 and the activator M18BP1 (Tetiker et al., in preparation). Using cryo-EM, single-molecule assays, and domain truncations, we are analyzing how these proteins influence condensin II’s activity. This multifaceted approach is intended to provide mechanistic insights into how condensin II is temporally and spatially regulated during the cell cycle.
Work package 3 focused on identifying the biological functions of loop extrusion. Specifically, we focused on the Wadjet system, a bacterial SMC complex composed of JetA–D, where JetA–C form a condensin-like motor module and JetD functions as a topology-dependent nuclease. Using biochemical reconstitution and single-molecule imaging, we demonstrated that Wadjet extrudes DNA loops symmetrically as a dimer on circular DNA and cleaves DNA upon completing loop extrusion and stalling (Pradhan et al., Mol. Cell, 2025). This is the first direct evidence for loop extrusion by a bacterial SMC complex and uncovers a novel role of loop extrusion in DNA-based immunity. The striking mechanistic parallels between Wadjet and eukaryotic Smc5/6—both of which protects host genome against covalently closed circular DNA—suggest an ancestral origin of SMC-based defense strategies.
In a separate study, we also revealed a previously unknown in vivo role of Smc5/6 in genome architecture. While Smc5/6 is established as a factor in DNA repair and resolution of chromatid linkages, its involvement in higher-order chromosome structure was unclear. Using a combination of single-molecule imaging, Hi-C, and ChIP-seq, we discovered that Smc5/6 is recruited to chromatin in response to transcription-induced positive supercoiling, where it forms cis-chromatin interactions and extrudes loops (Jeppsson et al., Mol. Cell, 2024). Single-molecule assays further showed that Smc5/6 preferentially loads at the tips of positively supercoiled DNA intertwines to initiate loop extrusion. These findings demonstrate that Smc5/6 functions in genome organization and uncover a molecular mechanism linking transcriptional activity to loop formation.
In work package 2, we aimed to investigate the regulatory mechanisms of SMC complexes. Our focus shifted from the well-characterized cohesin complex to the less-explored condensin I and II, aiming to understand their regulation by co-factors. For condensin I, we demonstrated that chromokinesin KIF4A regulates loop extrusion by binding to the HAWK subunit NCAPG in its C-terminal tail (Cutts et al., EMBO J., 2024). This interaction activates condensin I’s ATPase and loop extrusion activities, and notably, KIF4A alone is sufficient to trigger this activation. Extending these studies to condensin II, which is nuclear during interphase and activated during mitosis, we are investigating its regulation by candidate factors including the inhibitor MCPH1 and the activator M18BP1 (Tetiker et al., in preparation). Using cryo-EM, single-molecule assays, and domain truncations, we are analyzing how these proteins influence condensin II’s activity. This multifaceted approach is intended to provide mechanistic insights into how condensin II is temporally and spatially regulated during the cell cycle.
Work package 3 focused on identifying the biological functions of loop extrusion. Specifically, we focused on the Wadjet system, a bacterial SMC complex composed of JetA–D, where JetA–C form a condensin-like motor module and JetD functions as a topology-dependent nuclease. Using biochemical reconstitution and single-molecule imaging, we demonstrated that Wadjet extrudes DNA loops symmetrically as a dimer on circular DNA and cleaves DNA upon completing loop extrusion and stalling (Pradhan et al., Mol. Cell, 2025). This is the first direct evidence for loop extrusion by a bacterial SMC complex and uncovers a novel role of loop extrusion in DNA-based immunity. The striking mechanistic parallels between Wadjet and eukaryotic Smc5/6—both of which protects host genome against covalently closed circular DNA—suggest an ancestral origin of SMC-based defense strategies.
In a separate study, we also revealed a previously unknown in vivo role of Smc5/6 in genome architecture. While Smc5/6 is established as a factor in DNA repair and resolution of chromatid linkages, its involvement in higher-order chromosome structure was unclear. Using a combination of single-molecule imaging, Hi-C, and ChIP-seq, we discovered that Smc5/6 is recruited to chromatin in response to transcription-induced positive supercoiling, where it forms cis-chromatin interactions and extrudes loops (Jeppsson et al., Mol. Cell, 2024). Single-molecule assays further showed that Smc5/6 preferentially loads at the tips of positively supercoiled DNA intertwines to initiate loop extrusion. These findings demonstrate that Smc5/6 functions in genome organization and uncover a molecular mechanism linking transcriptional activity to loop formation.
The findings that resulted from the LoopSMC project significantly advance the fields of SMC complex biology, chromosome organization, and bacterial immunity. First, identifying Smc5/6 as an active loop extruder reshaped our understanding of its role in genome organization, changing its view beyond passive functions to a central, dynamic role comparable to cohesin and condensin. Given Smc5/6’s involvement in genome stability, transcription, and antiviral defense, this opens new avenues for exploring its multifunctionality via loop extrusion and highlights its evolutionary link to prokaryotic SMCs. Second, our discovery that the bacterial Wadjet complex extrudes DNA loops and restricts plasmids through dimerization-dependent, topology-sensitive mechanisms establishes loop extrusion as a evolutionarily conserved, fundamental principle of genome folding. Furthermore, this reveals a novel form of SMC-driven DNA immunity with implications for antiviral defense in eukaryotes. Finally, by uncovering that KIF4A regulates condensin I through SLiM-mediated interaction with condensin I’s HAWK subunit NCAPG, we demonstrate that SLiM–HAWK binding is a conserved mode of SMC complex regulation, offering new insights into condensin control and broader SMC regulatory mechanisms.