Periodic Reporting for period 4 - CondStruct (Structural basis for the coordination of chromosome architecture by condensin complexes)
Berichtszeitraum: 2020-04-01 bis 2021-12-31
The spatial and temporal arrangement of the DNA double helix is key to all functions of the genome. How the large-scale topology of chromatin fibers inside the eukaryotic nucleus is controlled and how this architecture changes during cell divisions to give rise to rod-shaped mitotic chromosomes have remained central unresolved questions of modern cell biology.
Mis-regulation of gene expression or unrestrained recombination events as a consequence of errors in the three-dimensional positioning of chromatin, as well as chromosome breakage, rearrangements, or aneuploidies because of a failure to condense chromosomes during mitosis or meiosis are hallmarks of various human disease conditions. Understanding the principles that maintain the integrity of genomes during health and how these change during disease is of immediate biomedical relevance for society.
During the recent years, multi-subunit protein complexes of the Structural Maintenance of Chromosomes (SMC) protein family have emerged as the central regulators of chromosome organization. The key goal of the ERC-funded research program was to uncover the molecular mechanisms that underlie the structural organization of chromosomes by the eukaryotic condensin complex as a paradigm for the role of SMC complexes in the preservation of eukaryotic genome architecture.
The research programme funded through this grant uncovered that SMC protein complexes are active motor proteins that use the energy of ATP hydrolysis to extrude DNA double helices into large loop structures. A highly interdisciplinary approach that integrated biochemistry, biophysics and structural biology techniques has provided near-atomic resolution insights into the molecular mechanisms that underlie the newly discovered DNA loop-extrusion activity. Together, these findings revolutionize our understanding of the fundamental principles that determine genome organization in all living species.
Mis-regulation of gene expression or unrestrained recombination events as a consequence of errors in the three-dimensional positioning of chromatin, as well as chromosome breakage, rearrangements, or aneuploidies because of a failure to condense chromosomes during mitosis or meiosis are hallmarks of various human disease conditions. Understanding the principles that maintain the integrity of genomes during health and how these change during disease is of immediate biomedical relevance for society.
During the recent years, multi-subunit protein complexes of the Structural Maintenance of Chromosomes (SMC) protein family have emerged as the central regulators of chromosome organization. The key goal of the ERC-funded research program was to uncover the molecular mechanisms that underlie the structural organization of chromosomes by the eukaryotic condensin complex as a paradigm for the role of SMC complexes in the preservation of eukaryotic genome architecture.
The research programme funded through this grant uncovered that SMC protein complexes are active motor proteins that use the energy of ATP hydrolysis to extrude DNA double helices into large loop structures. A highly interdisciplinary approach that integrated biochemistry, biophysics and structural biology techniques has provided near-atomic resolution insights into the molecular mechanisms that underlie the newly discovered DNA loop-extrusion activity. Together, these findings revolutionize our understanding of the fundamental principles that determine genome organization in all living species.
We first established different production pipelines for the recombinant expression and purification of multi-subunit condensin complexes from different species in their active form. Access to the ~0.7 MDa holo complexes and defined sub-complexes thereof subsequently enabled the in vitro study of their elemental properties and resolving their mode of action at near-atomic resolution.
Single-molecule imaging in collaboration with the groups of Prof. Cees Dekker (NL) and Prof. Eric Greene (USA) revealed that condensin complexes are DNA motor proteins that are capable of translocating along DNA helices over distances of several kilo-base pairs without turning around or falling off (Terakawa et al., Science 2017). Even more astonishing were the discoveries that condensin folds DNA into large loop structures while it moves along the DNA double helix (Ganji et al., Science 2018) and that it can do so while stepping over large obstacles, like other translocating condensins (Kim et al., Nature 2020). The first demonstration of DNA loop extrusion by an SMC protein complex provided vital proof for a new principle of genome organization, which had previously been predicted solely by simulations (discussed in Hassler et al., Curr Biol 2018).
How these large ring-shaped protein complexes extrude DNA loops was puzzling. Crystal structures of a condensin subcomplex revealed that it entraps the DNA double helix like a ‘safety belt’ holds on to a car passenger (Kschonsak et al., Cell 2017), thereby serving as an ‘anchor’ to enable the unidirectional extrusion of a DNA loop. Small angle x-ray scattering experiments with the group of Dr. Dmitri Svergun (DE) suggested that this anchor site specifically forms upon association of the two subunits (Manalastas-Cantos et al., J Biol Chem 2019). However, to move along DNA, condensin required an additional motor entity.
Crystal structures and biochemical characterization of the SMC ATPase domains uncovered an asymmetric ATPase reaction cycle that is created by distinct ATP binding and hydrolysis events at the two composite active sites (Hassler et al., Mol Cell 2019). Genomic engineering of the SMC ATPases in human cultured cells in collaboration with the group of Dr. Benjamin Rowland (NL) demonstrated that this asymmetric cycle is essential for the correct folding of mitotic chromosomes (Elbatsh et al., Mol Cell 2019).
The first cryo-electron microscopy structures of an SMC complex in its entity and at different functional states (Lee et al., Nat Struc Mol Biol 2020), obtained in in collaboration with the groups of Dr. Martin Beck (DE) and Dr. Jan Löwe (UK), put the ATPase reaction cycle into context and complemented additional co-crystal structures of the SMC ATPases with one of its HEAT-repeat subunits (Hassler et al., Mol Cell 2019). Together, these data suggested a ‘flip-flop’ mechanism that underlies the SMC DNA motor activity. In this model, the interface between the SMC ATPases and the HEAT-repeat subunit create a temporary DNA binding (‘motor’) site, in addition to the stable DNA anchor site at the other HEAT-repeat subunit (discussed in Datta et al., Curr Opin Struc Biol 2020). Measurements of condensin-mediated DNA compaction by magnetic tweezers, in collaboration with the group of Prof. Cees Dekker (NL), provided further support for the presence of two distinct DNA binding sites in the condensin complex (Eeftens et al., EMBO J 2017).
The first in-vitro reconstitution of condensin loading onto DNA and the first cryo-EM structures of condensin in the ATP-engaged state with DNA bound simultaneously at the ‘anchor’ and ‘motor’ sites finally provided the first comprehensive model to explain SMC-mediated DNA loop extrusion in atomic detail (Shaltiel et al., bioRxiv 2021). Single-molecule imaging experiments using condensin complexes with mutations that inactivated either ‘anchor’ or ‘motor’ DNA-binding sites functionally validated this model.
In summary, the work performed over the past 5.5 years as part of this action has provided the first direct proof for the organization of eukaryotic genomes by DNA loop-extruding molecular machines of the SMC protein family and revealed in-depth the mechanistic principles that underlie their action.
Single-molecule imaging in collaboration with the groups of Prof. Cees Dekker (NL) and Prof. Eric Greene (USA) revealed that condensin complexes are DNA motor proteins that are capable of translocating along DNA helices over distances of several kilo-base pairs without turning around or falling off (Terakawa et al., Science 2017). Even more astonishing were the discoveries that condensin folds DNA into large loop structures while it moves along the DNA double helix (Ganji et al., Science 2018) and that it can do so while stepping over large obstacles, like other translocating condensins (Kim et al., Nature 2020). The first demonstration of DNA loop extrusion by an SMC protein complex provided vital proof for a new principle of genome organization, which had previously been predicted solely by simulations (discussed in Hassler et al., Curr Biol 2018).
How these large ring-shaped protein complexes extrude DNA loops was puzzling. Crystal structures of a condensin subcomplex revealed that it entraps the DNA double helix like a ‘safety belt’ holds on to a car passenger (Kschonsak et al., Cell 2017), thereby serving as an ‘anchor’ to enable the unidirectional extrusion of a DNA loop. Small angle x-ray scattering experiments with the group of Dr. Dmitri Svergun (DE) suggested that this anchor site specifically forms upon association of the two subunits (Manalastas-Cantos et al., J Biol Chem 2019). However, to move along DNA, condensin required an additional motor entity.
Crystal structures and biochemical characterization of the SMC ATPase domains uncovered an asymmetric ATPase reaction cycle that is created by distinct ATP binding and hydrolysis events at the two composite active sites (Hassler et al., Mol Cell 2019). Genomic engineering of the SMC ATPases in human cultured cells in collaboration with the group of Dr. Benjamin Rowland (NL) demonstrated that this asymmetric cycle is essential for the correct folding of mitotic chromosomes (Elbatsh et al., Mol Cell 2019).
The first cryo-electron microscopy structures of an SMC complex in its entity and at different functional states (Lee et al., Nat Struc Mol Biol 2020), obtained in in collaboration with the groups of Dr. Martin Beck (DE) and Dr. Jan Löwe (UK), put the ATPase reaction cycle into context and complemented additional co-crystal structures of the SMC ATPases with one of its HEAT-repeat subunits (Hassler et al., Mol Cell 2019). Together, these data suggested a ‘flip-flop’ mechanism that underlies the SMC DNA motor activity. In this model, the interface between the SMC ATPases and the HEAT-repeat subunit create a temporary DNA binding (‘motor’) site, in addition to the stable DNA anchor site at the other HEAT-repeat subunit (discussed in Datta et al., Curr Opin Struc Biol 2020). Measurements of condensin-mediated DNA compaction by magnetic tweezers, in collaboration with the group of Prof. Cees Dekker (NL), provided further support for the presence of two distinct DNA binding sites in the condensin complex (Eeftens et al., EMBO J 2017).
The first in-vitro reconstitution of condensin loading onto DNA and the first cryo-EM structures of condensin in the ATP-engaged state with DNA bound simultaneously at the ‘anchor’ and ‘motor’ sites finally provided the first comprehensive model to explain SMC-mediated DNA loop extrusion in atomic detail (Shaltiel et al., bioRxiv 2021). Single-molecule imaging experiments using condensin complexes with mutations that inactivated either ‘anchor’ or ‘motor’ DNA-binding sites functionally validated this model.
In summary, the work performed over the past 5.5 years as part of this action has provided the first direct proof for the organization of eukaryotic genomes by DNA loop-extruding molecular machines of the SMC protein family and revealed in-depth the mechanistic principles that underlie their action.
The discovery that condensin complexes translocate along DNA double helix by using the energy of ATP hydrolysis has dramatically changed the conception of these ubiquitous protein complexes from passive chromosome scaffold components to active molecular motors. This discovery has been instrumental for the breakthrough demonstration of DNA loop extrusion by SMC complexes the following year. By now, DNA loop extrusion has been successfully reconstituted with different SMC protein complexes and hence it is has become clear that the folding of chromatin fibers into large loops is the fundamental principle that determines the structure and function of genomes in all kingdoms of life.
A second breakthrough accomplishment has been the determination of the structure of the entire ~0.7 MDa condensin protein complex by cryo-electron microscopy in different functional states and in complex with DNA. This achievement had been deemed close to impossible by some reviewers of the original grant proposal due to the inherent flexibility of the elongated complex and has moved beyond of what is currently considered state-of-the-art for cryo-electron microscopy.
A second breakthrough accomplishment has been the determination of the structure of the entire ~0.7 MDa condensin protein complex by cryo-electron microscopy in different functional states and in complex with DNA. This achievement had been deemed close to impossible by some reviewers of the original grant proposal due to the inherent flexibility of the elongated complex and has moved beyond of what is currently considered state-of-the-art for cryo-electron microscopy.