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Smc5/6 COMPLEX INFLUENCE THE 3D FOLDING OF CHROMOSOME: UNRAVELING THE DYNAMIC INTERPLAY OF CHROMATIN ORGANIZATION

 

Eukaryotic genomes are arranged in a linear fashion known to us as chromosomes. However, chromosomal DNA is longer and is hard to fit into the nucleus in its naked form, so it is compacted into chromatin fibres. This first compaction of chromatin is created through DNA wrapping around histone octamers or nucleosomes. Even above nucleosomes, several compactions are proposed like 30nm fibres. However, recent studies have suggested from molecular methodologies such as chromosome conformation capture (3C) and advanced microscopy, that chromatin folding above the nucleosome is a dynamic process that includes long-distance cis interactions (physical interactions between distant regions of the same chromosome, Cis interactions specifically refer to interactions that occur on the same chromosome), governing the folding of chromatin fibres on interface and mitosis of the cell cycle. So far the mechanisms driving these dynamic high-order folding remain unclear. 


Over the 25 years, several protein factors have been identified, functioning on mitotic chromosomes which have shown some of the long-distance interactions which are responsible for the high-order chromatin folding. These protein factors are ring-shaped, containing a trimeric core with a pair of SMC ATPases and a conserved kleisin, in addition to several additional regulatory subunits known as structural maintenance of chromosomes (SMC) complexes. 


Eukaryotes contain four SMC complexes: cohesin, condensin, the Smc5/6 complex, and the dosage compensation complex. These complexes are characterised by their structure. Besides the trimer, SMC complexes have various regulatory subunits rich in HEAT repeats (present in Huntingtin, Elongation factor 3, a subunit of protein phosphatase 2A, and TOR1).


Cohesin is primarily recognized for its role in maintaining the cohesion between sister chromatids. Condensin, which exists as two related complexes in vertebrates (condensin I and II), is chiefly acknowledged for its contribution to compacting and providing elasticity to chromosomes during mitosis. The dosage compensation complex is primarily associated with the formation of heterochromatin and the silencing of genes on the X chromosome in Caenorhabditis elegans. The Smc5/6 complex is primarily known for its involvement in DNA repair mechanisms and ensuring genome stability but its role is unclear.


The smc5/6 complex (Smc5/6) function has remained unclear but has been executed during late S- to G2/M-phase by preventing the formation of segregation-inhibiting chromatid linkages (physical connections between sister chromatids that prevent them from separating prematurely during cell division). However, Smc5/6 is also needed for DNA repair and control of homologous recombination. Interestingly, in Saccharomyces cerevisiae chromosomes, the association of Smc5/6 with chromosomes relies on cohesin, while the inactivation of Smc5/6 does not affect cohesin binding. Both complexes exhibit chromosomal presence during the early S-phase, with their strongest binding observed in the G2/M phase, and they are removed during anaphase. 


Through the studies eventually done this year, it has been identified that the Smc5/6 function is also been connected to DNA supercoiling and sister chromatid intertwining (SCIs). SCIs occur when the replication machinery rotates along with the DNA helix during DNA replication, causing the newly synthesized sister chromatids to become intertwined. On the other hand, DNA supercoiling arises when DNA unwinding occurs ahead of translocating polymerases or helicases, leading to overtwisting (positive supercoiling) and undertwisting (negative supercoiling) of the DNA helix. These supercoiling events can result in the formation of complex DNA structures known as plectonemic supercoils.


DNA supercoil and SCI are resolved by type I (Top1) and type II (Top2) topoisomerase enzymes by creating single and double-strand DNA breaks. However, both the topoisomerases can relax the supercoil, and only topoisomerase II can remove the SCI. So inhibition of Top2, during DNA replication, leads to the accumulation of SCI, while supercoiling got blocked on inhibition of both Top1 and Top2. Interestingly, Smc5/6, under normal conditions, its chromosomal association pattern is typically restricted to specific regions, such as centromeres. However, on Top2 inhibition during replication, Smc5/6 was no longer restricted to centromeres but was found in all regions between convergently oriented genes along S. cerevisiae chromosome arms. On the other hand, after replication, and inhibition of Top2 in G2/M-arrested cells, no change in the distribution of Smc5/6 was observed, suggesting the presence of direct and indirect association with SCI and Smc5/6. 


Additional experiments using magnetic tweezers and in vitro pull-down assays indicate that Smc5/6 can stabilize both positive and negative DNA supercoils and preferentially interact with positively supercoiled DNA. However, the exact mechanism by which Smc5/6 interacts with DNA supercoils and SCIs and how these interactions are linked to its function remains to be fully elucidated.


The recent discovery that the Smc5/6 complex acts as a DNA loop extruder in vitro prompted further investigation into its role in controlling the spatial organization of chromosomes within cells. This research uncovered a specialized function of Smc5/6 in organizing the genome, particularly in creating intrachromosomal links between specific chromosomal regions that undergo transcription-induced positive supercoiling.


Positive supercoiling occurs when DNA is overtwisted ahead of the transcription machinery during gene expression. These positively supercoiled regions are typically found at the base of cohesin-dependent DNA loops, which are structural features involved in chromosome organization. Interestingly, the study found that loop-extruding cohesin also plays a role in regulating the chromosomal binding of Smc5/6.


Single-molecule imaging techniques were employed to confirm that Smc5/6 preferentially binds to positively supercoiled DNA regions. Furthermore, the imaging revealed that dimers of Smc5/6 initiate the process of DNA loop extrusion specifically at the tips of positively supercoiled DNA structures known as plectonemes.


Overall, this research provides compelling evidence that all eukaryotic SMC complexes, including Smc5/6, play a crucial role in orchestrating the spatial organization of chromosomes within the nucleus. Additionally, it offers mechanistic insights into the cellular function of Smc5/6 and its intricate connections to other cellular processes such as cohesin-mediated loop formation, sister chromatid intertwinings (SCIs), and DNA supercoiling. These findings significantly advance our understanding of the molecular mechanisms governing chromosome organization and genome stability in eukaryotic cells.


REFERENCE


Aragón L. The SMC5/6 complex: New and old functions of the enigmatic long-distance relative. Annual Review of Genetics. 2018 Nov 23;52(1):89–107. doi:10.1146/annurev-genet-120417-031353 


Jeppsson K, Pradhan B, Sutani T, Sakata T, Umeda Igarashi M, Berta DG, et al. Loop-extruding SMC5/6 organizes transcription-induced positive DNA supercoils. Molecular Cell. 2024 Jan; doi:10.1016/j.molcel.2024.01.005 


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